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A 


SERUM  DIAGNOSIS  OF  SYPHILIS 
AND  LUETIN  REACTION 


TOGETHER   WITH 


THE  BUTYRIC  ACID  TEST 
FOR  SYPHILIS 


BT 

HIDEYO  NOGUCHI,  M.D.,  M.Sc. 

ASSOCIATE  MEMBER  OF  THE  KOCKEFELLER  INSTITUTE  FOR  MEDICAL  RESEARCH,  NEW  TOBK 


ILLUSTRATIONS,  OF  WHICH  17  ARE  IN  COLOR 


THIRD  EDITION 


PHILADELPHIA  &  LONDON 

J.  B.  LIPPINCOTT  COMPANY 


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Copyright,  1910 
By  J.  B.  Lippincott  Company 


Copyright,  1911 
By  J.  B.  Lippincott  Company 


Copyright,  1912 
By  J.  B.  Lippincott  Company 


Pv  C  ^  o  \ 


\^VL 


Printed  by  J.  B.  Lippincott  Company 
The  Washington  Square  Press,  Philadelphia,  U.S.A. 


To 
DR.  SIMON  FLEXNER 

THIS    BOOKLET    IS    DEDICATED    BY    HIS    PUPIL 
THE    AUTHOR 


Digitized  by  the  Internet  Arciiive 

in  2010  witii  funding  from 

Open  Knowledge  Commons 


http://www.archive.org/details/serumdiagnosisofOOnogu 


PREFACE  TO  THIRD  EDITION 

As  a  consequence  of  the  successful  cultivation  of 
Treponema  pallidum  in  artificial  media,  much  light 
has  been  thrown  on  the  nature  of  the  complement- 
fixation  reaction  in  syphilis.  With  the  aid  of  pure 
pallidum  cultures  the  writer  was  enabled  to  distin- 
guish the  Wassermann  reaction  from  the  specific  com- 
plement fixation  and  has  introduced  a  chapter  on  this 
question  in  the  present  edition.  By  establishing  this 
distinction  the  writer  hopes  to  develop  a  new  phase  of 
serum  diagnosis  in  this  disease. 

Another  feature  of  the  new  edition  is  the  addition 
of  a  chapter  on  the  luetin  reaction,  a  test  which  has 
been  put  to  trial  for  a  sufiicient  period  to  warrant 
a  detailed  description  for  those  who  are  interested  in 
this  phase  of  problems. 

As  for  the  technics,  no  alteration  has  been  found 
necessary  since  the  appearance  of  the  last  edition. 

The  writer  has  availed  himself  of  the  opportunity 
of  incorporating  certain  interesting  facts  revealed  by 
Craig  in  his  analysis  of  a  large  number  of  cases  treated 
with  salvarsan,  in  order  to  enrich  the  chapter  on  the 
effect  of  the  treatment  upon  the  serum  reaction. 

HiDEYO  NOGUCHI. 

Rockefeller  Institute  for  Medical  Research, 
New  York,  October,  1912. 


PREFACE  TO  SECOND  EDITION 

A  FEW  alterations  and  many  important  additions 
necessitated  by  the  growth  of  the  subject  have  been 
made  in  the  present  edition.  Instead  of  the  single 
method  of  performing  the  complement-deviation  test 
for  syphilis  described  in  the  first  edition  two  proced- 
ures are  given  in  order  to  widen  the  application  of  the 
test  under  various  conditions.  Both  methods  yield 
exactly  the  same  results. 

The  subject  of  the  syphilitic  antigen  has  been  al- 
most entirely  rewritten.  The  question  of  the  antigen 
has  long  been  one  of  the  most  difficult  problems  in 
the  whole  syphilis  reaction,  and  for  the  selection  of  a 
suitable  antigen  upon  which  much  of  the  reliability 
(and  a  wider  adaptability)  of  the  reaction  depends 
the  serologist  has  been  forced  to  resort  to  a  tedious 
empiricism  on  account  of  the  lack  of  a  definite  stand- 
ard. After  a  systematic  analytical  study  of  more 
than  one  hundred  organ  extracts  the  author  is  now 
able  to  supply  this  want  by  giving  a  definite  quantita- 
tive and  qualitative  standard  for  a  reliable  antigen. 
The  method  of  selection,  which  is  extremely  simple, 
and  the  best  mode  of  preservation  are  fully  described. 

A  technical  improvement  will  be  noticed  in  the 
method  for  quantitative  determination  of  the  inten- 
sity of  the  reaction;  and  the  results  of  its  application 


viii        PREFACE  TO  THE  SECOND  EDITION 

to  the  study  of  a  large  number  of  patients  treated  with 
the  Ehrlich-Hata  arsenobenzol  will  be  found  in  a 
later  chapter. 

In  the  chapter  dealing  with  the  clinical  value  of 
the  Wassermann  reaction  many  interesting  new  ob- 
servations made  by  Craig,  Kaliski,  Pedersen,  Fox, 
Atwood,  M.  Cohen,  Orleman-Robinson  and  others, 
have  been  introduced.  The  reliability  of  the  author's 
method  has  been  fully  confirmed  by  the  publications 
of  numerous  investigators,  comprising  a  study  of 
more  than  10,000  cases  of  syphilitic,  parasyphilitic 
and  other  diseases.  The  small  number  of  irregular 
results  obtained  by  one  or  two  early  workers  have 
been  adequately  accounted  for  by  their  failure  to 
observe  accurately  the  author's  directions.  These 
irregular  results  were  much  fewer  in  number  than 
those  obtained  with  the  original  Wassermann  reaction 
during  the  early  period  of  its  use. 

The  practical  value  of  the  author's  butyric  acid 
test  has  been  augmented  through  its  successful  appli- 
cation and  adoption  by  numerous  workers  in  psychi- 
atry and  neurology.  Flexner  has  employed  the  test 
as  an  aid  to  the  recognition  of  the  early  stage  of  acute 
anterior  poliomyelitis  in  man  and  in  experimentally 
infected  apes. 

Practically  all  of  the  literature  to  date  has  been 
incorporated  in  this  edition. 

HiDEYO  NOGUCHI. 
Febbxjart,  1911. 


PREFACE  TO  FIRST  EDITION 

Since  the  application  in  1906  by  Wassermann  and 
others  of  the  Bordet-Gengou  phenomenon  of  comple- 
ment fixation  to  the  diagnosis  of  syphilis,  the  large 
number  of  publications  on  the  subject,  while  establish- 
ing the  test  as  a  diagnostic  method  of  real  value,  have, 
unfortunately,  aided  the  general  medical  reader 
little  in  gaining  a  clear  conception  of  the  principles  in- 
volved ;  and  the  rapidly  growing  literature  has  become 
too  voluminous  to  enable  the  average  clinical  labora- 
tory worker  to  obtain  exact  data  on  the  test,  in  its 
various  forms. 

The  object  of  this  book  is,  first,  to  give  a  brief  yet 
adequate  account  of  the  principles  of  serum  haemol- 
ysis and  of  the  behaviors  of  the  combinations  of  anti- 
gens and  antibodies  towards  haemolysis,  so  essential 
for  a  proper  understanding  of  the  subject,  discussing 
at  some  length  the  quantitative  relationship  of  the  fac- 
tors playing  a  part  in  these  phenomena,  an  aspect  of 
the  subject  that  has  not  perhaps  received  the  consid- 
eration that  it  deserves;  and,  secondly,  to  give  in 
detail  the  technic  of  Wassermann's  method  and  of 
the  method  recommended  by  the  author. 

The  author  has  endeavored  to  treat  the  subjects  of 
this  book  in  such  a  manner  as  to  make  it  suitable  for 
the  use  of  practising  physicians  and  students,  and  at 


X  PREFACE  TO  FIRST  EDITION 

the  same  time  with  sufficient  comprehensiveness  to 
render  it  useful  to  laboratory  workers. 

The  last  chapter  is  a  digression  from  the  main 
subject,  being  devoted  to  a  description  of  a  chemical 
test  for  syphilis.  The  application  of  this  test  in  the 
examination  of  cerebrospinal  fluids  is  very  simple  and 
numerous  trials  have  shown  it  to  be  an  actual  aid  in 
the  diagnosis  of  parasyphilitic  affections  of  the 
central  nervous  system. 

In  the  appendix  the  author  has  given  an  extensive 
bibliography,  Avhich  should  be  of  value  to  readers  de- 
siring further  knowledge  on  the  subject  of  the  com- 
plement-fixation test  for  syphilis  and  certain  closely 
allied  problems  of  immunity,  as  well  as  on  the  cyto- 
logical  and  chemical  examination  of  cerebrospinal 
fluids,  so  useful  in  the  diagnosis  of  parasyphilitic 
conditions. 

The  author  wishes  to  express  his  indebtedness  to 
Dr.  Paul  Lewis  and  Dr.  David  J.  Kahski  for 
valuable  assistance  in  the  preparation  of  this  book. 
He  desires  also  to  thank  those  who,  by  furnishing 
specimens  for  examination,  facilitated  completion  of 
his  methods,  the  early  co-operation  of  Dr.  Victor  C. 
Pedersen  and  of  Dr.  J.  W.  Moore  having  been 
especially  valuable. 

HiDEYO  NOGUCHI. 


Rockefeller  Institute  for  Medical  Research, 
New  York,  February,  1910. 


CONTENTS 


CHAPTEE  PAGE 

I.     Serum  ILemoltsis 1 

II.     Quantitative  Facts  about  ILemoltsis 8 

III.  Antigens  and  Antibodies 17 

IV.  The  Application  of  the  Indirect  Method  of  Determining 

Antibodies  to  the  Diagnosis  of  Syphilis 26 

V.     Quantitative  Relations  of  the  Factors  in  the  Serum 

Diagnosis  of  Syphilis 31 

VI.     Various  Forms  of  the  Complement-Fixation  Test  as  Ap- 
plied TO  the  Serum  Diagnosis  of  Syphilis 36 

VII.     A  System  of  Serum  Diagnosis  of  Syphilis  Recommended 

BY  the  Author 50 

VIII.     Adjustability  of  the  Writer's  System 90 

IX.     Inactivation  of  the  Serum  in  Relation  to  the  Syphilis 

Reaction 95 

X.     Technic  of  the  Wassermann  System 102 

XI.  Diagnostic  Value  of  the  Serum  Reaction  of  Syphilis.  .  114 

XXL     The  Effect  of  Treatment  upon  the  Reaction 136 

XIII.  Specific  Complement-Fixation  in  Syphilis  159 

XIV.  The  Luetin  Reaction 167 

XV.    The  Butyric  Acid  Test 221 

Glossary 245 

Bibliography 253 

Index 299 

xi 


ILLUSTRATIONS 


PAQB 

Fig.  1.       Saline  suspension  of  blood-corpuscles  before  and  after  haemolysis; 

also  after  sedimentation  of  corpuscles 3 

Figs.  2,  3  and  4.     Amount  of  complement  needed  to  produce  a  given 

amount  of  haemolysis 14 

Fig.  5.  Components  required  and  steps  of  reaction  followed  in  producing 
haemolysis,  bacteriolysis,  and  adsorption  or  fixation  of  comple- 
ment by  a  precipitate . 22 

Fig.  6.       Illustrating  the  phenomenon  of  Bordet  and  Gengou 23 

Fig.  7.       Showing  manner  of  collecting  blood  with  a  Wright's  capsule ... .     55 

Figs.  8  and  9.     Showing  steps  necessary  in  sealing  the  Wright  capsule 

without  spoiling  the  blood  while  sealing  with  a  flame 56 

Fig.  10.     Appearance  of  all  tubes  after  first  incubation  and  just  at  the 

moment  when  the  amboceptor  slip  is  about  to  be  added 64 

Fig.  11.     Appearance  of  tubes  after  completion  of  haemolysis,  namely,  after 

second  incubation 65 

Fig.  12.     Appearance  of  tubes  after  standing  for  several  hours 66 

Fig.  13.     Showing  varying  degrees  of  inhibition  of  haemolysis 68 

Fig.  14.     Sterilize  in  steam,  but  not  in  hot  air 76 

CuEVE  1.     Heating  of  a  syphilitic  serum  to  different  tenperatures  for  20 

minutes  (in  a  water-bath) 97 

CxjEVE  2.    Heating  of  three  different  samples  of  syphilitic  sera  to  55°  C. 

for  varying  lengths  of  time  (in  a  water-bath) 98 

CtnavB  3.    Heating  of  a  serum  from  a  case  of  leprosy  to  55°  C 99 

xiii 


xiv  ILLUSTRATIONS 

PLATES. 

Plate  I.        Actively  immunized  rabbit  on  sixth  day  after  inoculation ....   172 

Plate  II.      Case  of  chancre,  followed  by  rupio-papular  eruptions 174 

Plate  III.    Hereditary  syphilis  and  interstitial  keratitis  of  six  months' 

standing 176 

Plate  IV.     Chancre,  followed  by  secondaries 178 

Plate  V.      Exostosis  and  stenosis  of  lacrimal  canal 180 

Plate  VI.     Typical  case  of  tardiac  congenital  syphilis  with  bilateral  inter- 
stitial keratitis,  Hutchinson  teeth,  saddle  nose,  etc 210 


Serum  Diagnosis  of 

Syphilis 

I. 

SERUM  HiEMOLYSIS. 

The  red  blood-corpuscles  of  animals  when  put 
in  contact  with  many  different  substances  are  so 
altered  that  their  haemoglobin  is  set  free,  the  strom- 
ata  also  going  into  solution,  as  a  rule.  This  phe- 
nomenon of  solution  is  now  generally  known  as 
hcemolysis.  The  substances  which  cause  haemolysis 
are  said  to  be  haemolytic  for  the  blood-corpuscles 
which  they  dissolve.  Fresh  blood-serum  of  many 
animal  species  is  heemolytic  for  the  erythrocytes  of 
some,  but  not  all,  other  species.  Hemolysis  by 
serum  results  from  the  cooperated  (coordinated)  ac- 
tion of  two  distinct  serum  principles  or  factors.  The 
first  is  called  amboceptor^  the  second  complement? 
The  latter  is  always  present  in  all  fresh  sera :  the 
former,  on  the  other  hand,  is  inconstantly  so,  fre- 
quently being  absent  from  normal  blood-serum.  If 
erythrocytes  are  added  to  a  serum  which  contains 
only  amboceptor  they  absorb  the  amboceptor  and 

^  Bordet's  substance  sensibilisatrice,  and  Metchnikoff's  fixateur. 
*  Bordet's  alexin,  and  Metchnikoff's  cytase. 

1 


2  SERUM  DIAGNOSIS  OF  SYPHILIS. 

retain  it  so  firmly  that  even  repeated  washing  with 
physiological  salt  solution  cannot  detach  it  from  the 
corpuscles.  The  erythrocytes  laden  with  ambocep- 
tor are  said  to  have  been  sensitized.  If  complement 
be  added  to  cells  so  prepared  they  promptly  dis- 
solve. Erythrocytes  do  not  absorb  complement 
from  a  serum  if  there  is  no  amboceptor  present. 
The  function  of  the  amboceptor  is  to  prepare  or 
sensitize  the  erythrocytes  for  the  attack  of  the  com- 
plement, and  that  of  the  complement  is  to  dissolve 
the  sensitized  erythrocytes.  Amboceptor  alone  can- 
not dissolve  the  cells  and  complement  likewise  is  in- 
effective if  the  cells  are  not  prepared  for  its  action. 
The  particular  constituent  of  the  erythrocytes 
capable  of  uniting  with  the  specific  amboceptor  is 
usually  called  the  receptor. 

A  suspension  of  erythrocytes  in  physiological  salt  solution  presents 
a  bright  orange-red,  opaque  appearance.  The  cells  may  be  sedimented 
to  the  bottom  of  the  receptacle,  either  by  centrifugalization  or  by  being 
allowed  to  stand  for  many  hours,  leaving  above  a  clear,  colorless  fluid. 
After  haemolysis,  however,  the  suspension  becomes  deep  pinkish  red  and 
transparent,  being  now  a  solution  of  haemoglobin  diffused  out  of  the 
haemolysed  erythrocytes.      (See  Fig.  1.) 

These  two  essential  heemolytic  components,  am- 
boceptor and  complement,  not  only  differ  in  their 
biological  function,  but  also  show  differences  in  re- 
sistance to  spontaneous  deterioration,  destruction  by 
heat,  and  various  other  physical  and  chemical  influ- 
ences.   Complement  is  liable  and  deteriorates  gradu- 


SERUM  DIAGNOSIS  OF  SYPHILIS. 


ally,  disappearing  from  serum  within  a  few  weeks 
when  kept  on  ice  and  within  a  few  days  when  kept 
at  room  temperature.  Exposure  to  a  temperature  of 
55° -56°  C.  for  one-half  hour  completely  destroys 
the  activity  of  complement.  Amboceptor  is  much 
more  stable.   It  is  usually  still  active  in  serum  which 


a 

b 

w 

fc^iii^ 

CL- 


Fig.  1. — a  shows  a  saline  suspension  of  blood-corpuscles  before  hsemolysis;  6,  the 
same  after  haemolysis,  a'  and  b'  present  the  appearance  of  a  and  6  after  sedimentation 
of  corpuscles. 

has  been  kept  for  more  than  a  year,  and  is  not 
destroyed  or  markedly  injured  by  exposure  to  a 
temperature  of  55° -56°  C.  Serum  is  technically 
known  as  "  fresh  "  or  "  active  "  serum  within  a  day 
of  its  collection,  while  the  complement  is  still  fully 
active.  The  process  of  depriving  a  fresh  serum  of 
its   complement  by  heating  it  to   55°    C.   is  called 


4  SERUM  DIAGNOSIS  OF  SYPHILIS. 

inactivation  of  the  serum.  By  an  inactivated  serum 
we  mean  one  from  which  complement  has  been  re- 
moved, but  in  which  the  amboceptor  is  left  un- 
changed. If  to  an  inactivated  serum  we  add  fresh 
serum  (in  a  quantity  inactive  by  itself  because  of 
the  minute  quantity  or  complete  absence  of  its 
amboceptor)  the  mixture  may  produce  haemolysis  in 
the  presence  of  amboceptor  in  the  inactive  serum, 
because  the  complement  destroyed  by  the  process  of 
inactivation  is  replaced  by  the  complement  of  the 
fresh  serum.  This  process  of  restoring  the  haemo- 
lytic  activity  of  an  inactivated  serum  by  the  addition 
of  fresh  serum  is  known  as  reactivation.  Fresh 
serum,  thus  used,  inactive  by  itself,  functionates  by 
virtue  of  its  complement-content,  and  is  commonly 
called  complement,  being  further  designed  by  the 
name  of  the  animal  from  which  it  is  derived.  Com- 
plement is  always  capable  of  reactivating  the  serum 
of  the  species  from  which  it  is  derived,  but  not  every 
complement  can  reactivate  the  sera  of  other  species. 
In  fact,  there  are  only  a  few  animals  known  whose 
complements  can  reactivate  the  inactivated  sera  of 
alien  species.  Otherwise  stated,  complement  of  one 
animal  species  is  not  identical  in  its  action  with  that 
of  another  species.  The  interchangeability  of  com- 
plements, or  the  substitution  of  one  for  that  of  another 
species  of  animal,  is  only  possible  in  a  limited  number 


SERUM  DIAGNOSIS  OF  SYPHILIS.  5 

of  instances.  The  complement  of  the  guinea-pig  is 
distinguished  by  an  unusual  ability  to  reactivate  the 
sera  of  alien  species  and  is  consequently  most  often 
used  when  it  is  necessary  to  substitute  the  complement 
of  one  serum  for  that  of  another  which  has  been  in- 
activated or  which  has  deteriorated. 

The  presence  of  amboceptor  in  blood-serum  is 
much  less  constant  than  that  of  complement. 

The  amboceptor  of  any  species  acts  always  with 
the  complement  of  the  same  species  and  less  regularly, 
and  to  a  limited  extent  only,  with  the  complement  of 
other  species.  In  its  relation  to  the  red  blood-cor- 
puscles, however,  the  amboceptor  is  specific:  that  is, 
an  amboceptor  which  can  sensitize  the  erythrocytes  of 
the  rabbit,  for  example,  to  the  action  of  complement 
cannot  sensitize  the  erythrocytes  of  the  sheep,  dog, 
or  any  other  animal.  Amboceptors  are  named  by 
prefixing  "  anti-  "  to  the  species-name  of  the  cells 
against  which  they  act.  Thus  an  amboceptor  active 
against  sheep-corpuscles  is  known  as  antisheep  am- 
boceptor, and  unites  with  the  receptors  of  the  former. 

In  any  serum  there  are  usually  present  many 
varieties  of  amboceptor.  Thus  in  one  serum  there 
may  be  found  amboceptors  active  against  the  blood- 
corpuscles  of  the  sheep,  dog,  hen,  rabbit,  frog,  man, 
etc.  Different  sera  vary  widely  in  the  number  of 
amboceptors  present  and  in  the  relative  quantity  of 


6  SERUM  DIAGNOSIS  OF  SYPHILIS. 

each.  Among  different  specimens  of  serum  from  the 
same  species  the  relative  quantity  of  amboceptor  can 
also  vary  considerably. 

Amboceptors  existing  naturally  in  normal  serum 
are  known  as  natural  or  normal  amboceptors.  For 
example,  human  serum  is  frequently,  though  not  al- 
ways, quite  hsemolytic  for  sheep's  corpuscles,  because 
it  may  contain  natural  antisheep  amboceptor;  but 
rabbit's  serum,  on  the  other  hand,  is  incapable  of 
hasmolysing  human  erythrocytes  because  of  the  ab- 
sence in  the  rabbit  of  natural  antihuman  amboceptor. 

As  already  stated,  the  serum  of  a  given  species 
may  not  contain  amboceptors  for  the  blood-corpuscles 
of  some  other  species.  If  we  select,  for  example,  the 
rabbit,  whose  blood-serum  contains  no  amboceptor 
for  the  erythrocytes  of  man,  we  may  by  repeated  in- 
jections of  the  human  erythrocytes  into  this  animal 
produce  specific  amboceptor  for  human  cells.  This 
process  of  repeated  injections  with  foreign  cells  (or 
any  other  suitable  substance)  is,  in  general,  known  as 
immunization.  By  a  similar  process  we  can  also  in- 
crease the  amount  of  an  amboceptor  naturally  pres- 
ent. Amboceptors  thus  artificially  produced  or  in- 
creased are  known  as  immune  amboceptors.  Ambo- 
ceptors which  act  with  the  erythrocytes  of  the  same 
species  are  known  as  isohcemolytic  amboceptors,  or,  in 
short,  isohcemolysins.    It  is  extremely  difficult  to  pro- 


SERUM  DIAGNOSIS  OF  SYPHILIS.  7 

duce  an  amboceptor  for  the  erythrocytes  of  the  same 
species  by  immunization. 

The  amount  of  complement  is  not  perceptibly 
increased  by  immunization. 

Summary. — We  have  learned  that  the  fresh  serum 
of  an  animal  can  hsemolyse  the  er5i:hrocytes  of 
another  species  or  of  as  many  species  as  its  serum  con- 
tains specific  amboceptors  for.  The  intensity  of 
haemolysis  is,  of  course,  proportional  to  the  amount 
of  each  amboceptor  present  in  the  serum.  The  variety 
of  natural  amboceptors  varies  considerably  accord- 
ing to  the  animal  species. 

The  haemolysis  is  caused  by  the  specific,  coordi- 
nated interaction  of  the  amboceptor  and  complement 
of  the  serum  on  the  erythrocytes.  Complement  is  de- 
stroyed by  heating  to  55°-56°  C.  for  half  an  hour,  the 
serum  being  thereby  inactivated.  Amboceptor  is  not 
destroyed  by  this  process.  The  hemolytic  activity 
of  the  serum  can  be  restored  by  replacing  the  de- 
stroyed complement  by  the  addition  of  complement 
contained  in  fresh  serum  of  the  same  species  or  in  that 
of  a  limited  range  of  alien  species  whose  complements 
are  suitable  for  use.  By  immunization  we  can  create 
a  specific  amboceptor  for  any  kind  of  foreign  eryth- 
rocytes. This  immunization  product  possesses  the 
same  biological  properties  as  the  natural  amboceptor 
and  is  called  immune  amboceptor. 


II. 

QUANTITATIVE  FACTS  ABOUT  HEMOLYSIS. 

The  phenomenon  of  heemolysis,  as  pointed  out 
in  the  previous  chapter,  is  dependent  upon  the  action 
of  complement  and  amboceptor  upon  erythrocytes. 
The  first  is  contained  in  every  fresh  serum,  the  second 
may  be  contained  in  a  given  serum  or  can  be  artifi- 
cially produced  by  immunization. 

The  hsemolytic  activity  of  any  serum  is  deter- 
mined by  mixing,  in  a  series  of  test-tubes,  a  uniform 
quantity  of  a  suspension  of  red  corpuscles  (erythro- 
cytes )  in  a  physiological  salt  solution  ^  with  graduated 
amounts  of  serum  and  bringing  the  whole  to  a  con- 
stant volume  by  the  addition  of  salt  solution.  The 
mixtures  are  placed  at  a  temperature  of  37°  C.  for 
a  sufliciently  long  time  to  allow  a  complete  reaction. 
The  titre  of  the  serum  is  usually  expressed  by  the 
smallest  amount  of  serum  which  is  found  to  be  neces- 
sary for  the  complete  dissolution  of  all  the  corpuscles. 
With  ordinary  normal  serum,  complement  is  usually 
present  in  excess  of  the  amount  needed  to  activate 
all  the  amboceptor  contained  in  the  serum.    The  titre 

^For  haemolytic  work  0.85  per  cent.  (Ehrlich)  to  0.9  per  cent. 
(Madsen)  salt  solution  is  universally  employed.  The  writer  uses  the 
latter  concentration. 


SERUM  DIAGNOSIS  OF  SYPHILIS.  9 

of  the  serum  in  this  case  can  be  readily  obtained  by 
the  above  method ;  and  it  is  dependent  entirely  on  the 
amount  of  amboceptor  present,  for,  as  will  be  pointed 
out  later,  the  activity  of  amboceptor  is  fully  revealed 
only  in  the  presence  of  an  excess  of  complement.  The 
conditions  in  dealing  with  an  immune  serum  are  more 
complicated.  In  it  the  normal  complement-content 
is  found  associated  with  a  great  excess  of  amboceptor. 
A  titration  by  the  above  method  of  simple  dilution 
would  disclose  only  the  amount  of  complement  in  the 
serum,  and  a  large,  variable  amount  of  amboceptor 
would  remain  inactive  in  the  mixture,  on  account  of 
the  dilution  to  a  minimum  of  complement.  In  order 
to  arrive  at  the  value  of  the  serum  in  terms  of  its 
amboceptor  another  procedure  must  be  adopted. 
There  is  placed  in  a  series  of  test-tubes,  as  before,  a 
definite  and  equal  amount  of  corpuscle  suspension 
and  to  each  tube  is  then  added  an  amount,  also  definite 
and  equal,  of  a  normal  serum  which  has  been  found 
incapable  in  itself  of  causing  haemolysis  (complement, 
see  Chapter  I ) .  There  are  next  added,  in  series,  de- 
creasing, graduated  amounts  of  the  immune  serum 
whose  native  complement  has  been  destroyed  by  in- 
activation.  The  titre  of  the  immune  serum  is  usually 
stated  as  the  smallest  amount  of  inactivated  serum 
which  produces  complete  haemolysis  in  the  presence 
of  an  excessive  amount  of  a  given  suitable  comple- 


10  SERUM  DIAGNOSIS  OF  SYPHILIS. 

ment.  It  must  be  borne  in  mind  that  the  titre  of  an 
immune  serum  will  vary  with  the  specific  activity  of 
the  complement  used.  For  example,  an  antihuman 
amboceptor  prepared  by  immunizing  a  rabbit  with  hu- 
man corpuscles  is  several  times  more  active  in  the  pres- 
ence of  a  given  amount  of  guinea-pig  serum  used  as 
complement  than  it  is  when  tested  with  the  same  quan- 
tity of  human  serum  as  complement.  The  strength  of 
the  immune  serum  expressed  in  terms  of  the  smallest 
amount  needed  to  produce  complete  haemolysis  will 
be  quite  different  if  0.1  c.c.  of  complement  has  been 
used,  from  the  value  found  if  0.05  c.c.  has  been  used. 
Within  certain  limits  the  quantitative  relationship 
existing  between  the  absolute  amount  of  comple- 
ment and  amboceptor  required  to  produce  complete 
hcemolysis  is  such  that  an  increase  of  one  factor, 
say  complement,  permits  the  use  of  a  less  amount  of 
the  other  factor,  namely  the  amboceptor. 

In  order  to  get  uniform  results  with  a  reaction  in 
which  so  many  of  the  reagents  are  variable  in  activity, 
it  is  necessary  to  proceed  in  a  definite  order  to  fix 
standards  of  practical  constancy.  This  order  has  been 
roughly  outlined,  but  will  bear  restatement.  A  suit- 
able suspension  of  erythrocytes  is  chosen  and  the 
amount  of  this  suspension  to  be  used  arbitrarily  fixed 
and  kept  constant.  The  total  volume  of  the  mixture 
is  decided  upon  as  another  constant.     A  large  and 


SERUM  DIAGNOSIS  OF  SYPHILIS.  11 

uniform  amount  of  complement  is  used  in  the  first 
determination,  an  amount  certainly  in  excess  of  that 
required  for  the  complete  activation  of  the  minimal 
amount  of  amboceptor.  With  this  amount  of  com- 
plement, in  series,  are  combined  decreasing  quantities 
of  amboceptor.  The  smallest  amount  of  amboceptor 
required  to  produce  complete  hsemolysis  is  determined. 
This  is  not  only,  as  was  before  stated,  the  titre  or  value 
of  the  immune  serum,  but  it  is  best  chosen  for  future 
work  as  the  second  fixed  value  in  the  reaction,  the 
erythrocyte  suspension  being  the  first.  In  order  to  be 
sure  that  this  is  a  constant  value  another  test  should 
now  be  made  in  which  the  quantity  of  amboceptor  is 
still  further  reduced  while  the  complement  quantity 
is  doubled.  If  all  of  the  tubes  in  this  series  are  not 
completely  hgemolysed  we  may  be  certain  that  the  com- 
plement quantity  first  chosen  was  large  enough  and 
that  the  amboceptor  amount  determined  was  actually 
the  least  amount  which  could  under  any  circumstances 
produce  complete  haemolysis  of  the  corpuscle  suspen- 
sion used.  This  amount  of  amboceptor  may  be  con- 
veniently designated  as  one  amboceptor  unit. 

Up  to  this  point,  it  will  be  recalled,  the  amount  of 
complement  has  been  kept  in  excess.  Now  to  each  of 
a  series  of  tubes  containing  the  standard  erythrocyte 
suspension  one  unit  of  amboceptor  is  added.  If  then 
in  the  series  one  puts  decreasing  amounts  of  comple- 


12  SERUM  DIAGNOSIS  OF  SYPHILIS. 

meiit  the  smallest  amount  of  complement  necessary 
to  produce  complete  haemolysis  with  one  amboceptor 
unit  will  be  determined.  This  amount  is  called  one 
complement  unit.  The  reaction  could  then  be  formu- 
lated as  follows: 

Standard  erythrocj'^te  suspension  +  1  amboceptor 
unit  +  1  complement  unit  =  complete  haemolysis 
(time  2  hours  at  37°  C). 

As  the  erythrocyte  suspension  can  be  made  of 
relatively  constant  value  from  day  to  day  and  as  the 
amboceptor  is  stable  over  a  period  of  months,  the 
only  actual  variable  from  this  point  is  the  complement, 
and  this  can  be  standardized  with  a  simple  titration 
test. 

The  results,  now,  of  varying  the  quantities  of  com- 
plement and  amboceptor  require  the  most  careful 
consideration.  If  less  than  one  unit  of  amboceptor 
is  used  haemolysis  will  always  be  incomplete,  even 
with  more  than  one  unit  of  complement.  Likewise 
if  with  one  amboceptor  unit  there  is  combined  less 
than  one  unit  of  complement,  haemolysis  cannot  be 
complete.  If  with  more  than  one  unit  of  amboceptor 
there  be  used  less  than  one  unit  of  complement, 
haemolysis  may  be  complete  or  incomplete  according 
to  the  relative  amounts  of  each  factor  used.  The 
complement  may,  of  course,  be  reduced  to  such  a 
degree  that  haemolysis  will  be  incomplete  no  matter 


SERUM  DIAGNOSIS  OF  SYPHILIS.  13 

how  much  amboceptor  is  present.  But  in  the  pres- 
ence of  many  units  of  amboceptor  haemolysis  may  he 
complete  when  hut  a  small  fraction  of  the  complement 
unit  is  present.  It  is  most  important  to  note  that  a 
fraction  of  one  unit  of  complement,  too  weak  to  pro- 
duce any  hsemolysis  with  one  unit  of  amboceptor,  can 
produce  complete  hsemolysis  when  combined  mth 
several  amboceptor  units.  In  other  words,  the  activity 
of  complement  becomes  steadily  intensified  or  aug- 
mented by  gradual  increase  of  the  number  of  ambo- 
ceptor units  until  its  maximum  is  reached.  An 
amount  of  complement  too  small  to  cause  complete 
hsemolysis  in  combination  with  one  unit  of  ambo- 
ceptor, may  be  sufficient  to  do  so  if  two  units  of  am- 
boceptor are  used,  and  an  inactive  quantity  of  com- 
plement with  two  units  of  amboceptor  may  no  longer 
be  inactive  when  four  units  or  more  are  used,  and  so 
forth.  Based  upon  this  fundamental  law  of  reduction 
in  requirement  of  complement  by  increase  of  ambo- 
ceptor to  cause  the  same  degree  of  hsemolysis,  we  can 
easily  see  that  hsemolysis  is  merely  the  relative  ex- 
pression of  the  combined  action  of  amboceptor  and 
complement  and  is  not  the  absolute  indication  of  the 
amount  of  the  hsemolytic  components  present  in  a 
fluid.  The  same  amount  of  hsemolysis  can  be  pro- 
duced by  one  unit  of  complement  and  one  unit  of 
amboceptor  or  by  20  units  of  amboceptor  and  0.1  unit 


14  SERUM  DIAGNOSIS  OF  SYPHILIS. 

of  complement,  or  any  other  appropriate  combinations 
of  these  two  components.  Unless  the  amount  of  am- 
boceptor used  is  the  same  in  any  two  sets  of  hcemolytic 
experiments  the  amount  of  complement  acting  in 
these  two  sets  cannot  he  estimated  by  comparing  the 
degree  of  hcemolysis.  In  order  to  calculate  the  exact 
amount  of  complement  needed  to  produce  a  given 
amount  of  haemolysis  it  is  necessary  to  know  the 
amount  of  amboceptor  used,  because  the  activity  of 
complement  is  different  according  to  the  amount  of 
amboceptor  present.  It  is  therefore  erroneous  to  con- 
clude that  equal  degree  of  haemolysis  produced  by  one 
unit  of  amboceptor  and  by  twenty  units  of  ambocep- 
tor is  the  work  of  the  same  amount  of  complement  in 
both  instances.  These  facts  are  graphically  shown  in 
the  accompanying  diagrams  (Figs.  2,  3  and  4). 

For  any  given  relative  proportion  the  result  can 
be  determined  experimentally,  of  course,  and  the  re- 
action follows  certain  laws  with  sufficient  regularity 
so  that  the  result  can  be  calculated.  But  it  is  ex- 
actly for  the  purpose  of  avoiding  the  complexities 
and  uncertainties  introduced  by  this  quantitative 
variation  that  we  take  such  pains  to  get  fixed  stand- 
ards, and  the  more  closely  these  standards  are  adhered 
to  the  simpler  will  be  the  conditions  and  the  more  easy 
and  accurate  the  interpretation  of  the  results. 

We  will  now  consider  the  fate  of  amboceptor  and 


2o  units  of^  Ambocepfor 
used  /n  e<3c/i  como/narion 


Green  ■=  Complement 
Purple  =  Amboceptor 
Red    =•  /isemolyiis 


I 


t'.:- 

<$ 

\ 

^ 

'O 

% 

^ 

V 

^ 

1- 

4 

s 

^ 

^ 

)i« 

^ 

<a 

5 

f  ^- 

^ 


I  unit  of  Amboceptor 
used  in  eacli  mf/i  Msnous 
frdctions  of- a  complement 
unit 


S  S  ■'5 


Fig.  2. 


Fig.  3. 


Fig.  4. 


SERUM  DIAGNOSIS  OF  SYPHILIS.  15 

complement  after  the  reaction  has  run  to  completion. 

It  has  been  experimentally  determined  that  with- 
in certain  quantitative  limits  they  have  disappeared 
from  the  mixture,  having  been  used  up  in  producing 
hemolysis.  The  limits  within  which  this  disappear- 
ance is  complete  are  roughly  as  follows:  Where  one 
unit  of  amboceptor  has  been  used  with  one  unit  of 
complement  the  disappearance  of  both  will  be  com- 
plete. When  with  one  unit  of  amboceptor  more  than 
one  unit  of  complement  is  used,  an  excess  of  comple- 
ment will  be  found  still  present  in  the  fluid  after  the 
reaction  is  accomplished.  The  same  applies  to  the 
combination  of  one  unit  of  complement  with  less  than 
one  unit  of  amboceptor,  in  which  case  haemolysis  will 
be  incomplete.  When  with  one  unit  of  complement 
there  is  combined  more  than  one  unit  of  amboceptor 
the  excess  of  amboceptor  shortens  the  time  necessary 
for  complete  haemolysis.  The  corpuscles  being 
capable  of  absorbing  more  amboceptor  than  is 
necessary  for  complete  haemolysis,  most  of  the  excess 
is  taken  up  by  them  in  this  way.  If  the  excess  is  very 
large  (more  than  the  corpuscle  mass  can  absorb), 
it  may  be  found  free  in  the  fluid  after  the  reaction 
is  ended.  If  more  than  one  unit  of  amboceptor  and 
of  complement  are  used  an  excess  of  both  may  be 
found  in  the  fluid  after  the  haemolysis  is  complete. 

Enough  has  been  stated  to  show  the  reader  that 


16  SERUM  DIAGNOSIS  OF  SYPHILIS. 

we  are  dealing  with  a  complex  reaction  whose  factors 
have  a  definite  relationship  to  one  another  which  can 
be  accurately  determined.  If  the  co-relative  values 
of  complement  and  amboceptor  are  carefully  deter- 
mined and  rigidly  adhered  to  according  to  the  outline 
given,  precise  and  constant  results  can  be  obtained  in 
the  practical  apphcation  of  the  reaction,  which  will 
be  developed  in  the  following  pages.  If  in  using  the 
reaction  these  quantitative  steps  are  not  observed  one 
cannot  hope  for  useful  or  accurate  results. 


III. 

ANTIGENS   AND    ANTIBODIES. 

I  HAVE  already  stated  that  by  repeated  injections 
of  erythi'ocytes  of  one  animal  into  an  alien  species  we 
can  produce  in  the  latter  an  amboceptor  having  a 
specific  affinity  towards  these  erythrocytes.  A  simi- 
lar phenomenon  is  observed  when  bacteria  are  in- 
jected. We  call  amboceptors  for  the  blood-corpuscles 
hcEmolytic  amboceptors  and  those  for  the  bacteria 
bacteriolytic  amboceptors.  Their  general  character- 
istics are  the  same,  differing  only  in  that  they  have  a 
specific  affinity  towards  the  substances  which  gave  rise 
to  them.  Bacteria  when  brought  in  contact  with  spe- 
cific bacteriolytic  amboceptor  absorb  the  latter  and  be- 
come sensitive  to  the  dissolving  action  of  complement. 
This  phenomenon  of  dissolution  is  called  bacteriolysis 
and  in  its  mechanism  is  comparable  to  that  of 
hcemolysis  in  every  respect. 

It  is  found  that  when  various  unorganized  protein 
substances  are  injected  into  an  animal  they  elicit  a 
similar  response,  giving  rise  to  various  specific  im- 
mune substances  or  reaction  products.  Egg  albumin 
and  alien  blood-serum,  for  example,  when  injected 
give  rise  to  specific  precipitins.     Serum  containing  a 

17 


18  SERUM  DIAGNOSIS  OF  SYPHILIS. 

specific  precipitin  when  mixed  with  a  solution  of  the 
protein  which  was  injected  in  order  to  develop  it 
causes  a  precipitate  to  form.  Mixed  with  any  other 
protein  solution  the  precipitation  does  not  occur. 
The  substances — erythrocytes,  bacteria,  or  unorgan- 
ized foreign  protein — which  when  injected  produce 
corresponding  specific  reaction  products — are  called 
antigens.  The  reaction  products  of  whatever  kind 
which  are  produced  by  the  animal  are  called  anti- 
bodies. Immune  haemolytic  amboceptors,  bacteriolytic 
amboceptors,  and  precipitins  are  therefore  antibodies 
produced  by  injecting  as  antigens  erythrocytes, 
bacteria,  or  unorganized  proteins. 

The  most  striking  characteristic  of  the  antibodies 
is  their  specific  relationship  with  the  corresponding 
antigens.  Antigen  A  gives  rise  to  antibody  A,  and 
antibody  A  reacts  outside  the  body  with  antigen  A 
and  with  no  other.  It  is  scarcely  necessary  to  recall 
the  w^ell-known  fact  that  the  serum  of  a  typhoid 
patient  (containing  typhoid  agglutinin)  agglutinates 
only  the  typhoid  bacillus  and  none  of  the  closely  re- 
lated intestinal  micro-organisms.  The  hsemolytic  am- 
boceptor for  human  erythrocytes  acts  only  with  those 
erythrocytes,  and  not  with  the  cells  of  any  other 
species.  Similarly,  as  was  before  stated,  the  precipitin 
obtained  by  injecting  man,  monkey,  or  rabbit  serum 
causes  precipitation  only  with  the  particular  serum 


SERUM  DIAGNOSIS  OF  SYPHILIS.  19 

used  to  produce  it.    The  few  exceptions  to  this  general 
rule  may  be  neglected  in  our  present  discussion.^ 

Taking  into  account  this  quality  of  specificity  it 
will  be  readily  seen  that  if  we  have  an  unknown  anti- 
body to  deal  with  we  can  identify  it  by  putting  it  in 
contact  with  a  number  of  different  antigens  under 
favorable  conditions  and  noting  the  one  with  which  it 
reacts.  With  a  known  antibody  the  character  of  an 
unknown  antigen  can  likewise  be  determined.  This 
direct  method  of  recognizing  unknown  antibody  has 
been  used  in  a  number  of  different  ways  in  recent 
years.  Some  instances,  such  as  the  Widal  reaction  in 
typhoid  fever,  are  known  to  every  one.  Another  im- 
portant test  dependent  on  this  principle  is  the  precipi- 
tation method  for  determining  the  species  of  animal 
from  which  a  specimen  of  blood  of  unknown  origin 
may  have  come.  Artificial  antibodies  are  produced  hy 
immunizing  animals — rabbits,  for  example — ^with  the 
blood-serum  of  a  number  of  different  animal  species. 
The  unknown  blood  is  dissolved  in  physiological  salt 
solution  and  put  in  contact  with  this  series  of  known 
antibodies  ( precipitins ) .  That  antibody  with  which 
a  precipitate  is  formed  must  be,  according  to  the  law 

^  For  example,  I  found  that  antigoat  serum  (rabbit)  contains  pre- 
cipitins not  only  for  goat  serum  but  also  for  sheep  and  ox  sermns 
in  smaller  quantity,  while  anti-ox  serum  (rabbit)  contains  precipitin  only 
for  ox  serum.  Antisheep  serum  contains  precipitin  for  sheep  and  goat 
serums,  but  not  for  ox  serum.  This  phenomenon  is  comparable  to  group 
reaction  of  agglutination. 


20  SERUM  DIAGNOSIS  OF  SYPHILIS. 

of  specificity,  antibody  prepared  with  serum  of  the 
same  species  as  that  from  which  the  specimen  in  ques- 
tion was  derived. 

The  effects  we  have  so  far  considered  have  all  been 
the  direct  and  essential  manifestation  of  reactions  be- 
tween antigen  and  antibody  with  or  without  the  asso- 
ciated action  of  complement  as  the  case  may  be. 

Studying  the  phenomena  of  interaction  of  antigen 
and  antibody  in  general,  we  will  discover  a  peculiar 
relation  which  exists  between  the  combination  of  an- 
tigen-antibody and  complement.  We  have  already 
seen  that  the  erythrocytes  (antigen)  acted  on  by  am- 
boceptor (antibody)  become  so  altered  as  to  absorb 
complement  and  undergo  haemolysis.  We  have  also 
learned  that  bacteria  (antigen),  after  having  been 
acted  on  by  amboceptor  (antibody),  take  up  com- 
plement and  become  dissolved  by  the  complement. 
Now  a  question  arises  as  to  whether  unorganized 
antigens  display  the  same  characteristics  as  the  anti- 
gens in  previous  instances  when  brought  together 
with  their  specific  antibodies.  Through  experiment 
this  was  found  to  be  the  case.  Thus  when  a  precipi- 
table  antigen  (precipitinogen)  is  brought  in  contact 
with  its  specific  precipitin  it  not  only  forms  a  visible 
precipitate,  but  also  becomes  capable  of  absorbing  or 
fixing  complement.  If  to  a  mixture  of  a  precipitable 
antigen — for  example,  blood-serum  or  egg  albumin — 


SERUM  DIAGNOSIS  OF  SYPHILIS.  21 

and  its  precipitin,  complement  be  added  during  or 
after  the  reaction  period,  and  if  the  mixture  be  sub- 
sequently tested  for  the  presence  of  complement  by 
adding  erythrocytes  and  their  specific  hsemolytic  am- 
boceptor to  the  mixture,  it  is  found  that  the  comple- 
ment has  disappeared ;  that  is,  hsemolysis  does  not  take 
place.  This  phenomenon  of  disappearance  of  com- 
plement in  the  mixture  of  antigen  and  antibody  is 
now  generally  called  fixation  of  complement.  Some- 
times it  is  called  deviation  of  complement  on  account 
of  the  fact  that  the  complement  has  been  deviated  by 
the  combination  of  antigen  and  antibody  and  pre- 
vented from  participating  in  the  hsemolytic  process. 
These  facts  were  first  brought  out  by  the  investiga- 
tions of  Bordet  and  Gengou  and  the  reaction  is  known 
accordingly  as  the  Bordet-Gengou  phenomenon  of 
complement  fixation. 

It  is  found  that  the  mixture  of  antigen  and  anti- 
body can  fix  complement  in  a  dilution  in  which  a 
visible  precipitation  is  no  more  obtainable.  In  other 
words,  the  fixation  phenomenon  is  capable  of  indicat- 
ing the  existence  of  the  antigen-antibody  reaction 
beyond  the  delicacy  that  a  visible  precipitation  can 
attain. 

Concerning  the  phenomenon  of  complement  fixa- 
tion, it  would  be  well  to  point  out  here  that  the  sera 
( complement )  of  various  animals  present  marked  dif - 


SERUM  DIAGNOSIS  OF  SYPHILIS. 


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24  SERUM  DIAGNOSIS  OF   SYPHILIS. 

ferences  in  regard  to  this  property.  Some  comple- 
ments are  easily  fixed  in  the  presence  of  the  antigen- 
antibody  combination,  others  slightly  or  not  at  all. 
While  the  serum  of  an  animal  may  possess  the  prop- 
erty of  reactivating  the  hfemolytic  amboceptor  of  an 
inactivated  serum,  yet  the  serum  of  this  species  may 
possess  little  or  no  fixation  property.  This  fact  be- 
comes of  great  importance,  as  will  be  seen  later,  in 
utilizing  the  complement-fixation  phenomenon  in 
diagnosis. 

Working  with  three  different  antigen-antibody 
combinations,  namely,  precipitates  formed  by  mixing 
human  serum,  egg  albumin,  and  meningococcus  ex- 
tract with  their  specific  precipitins,  the  writer  has 
found  that  the  fixability  of  the  sera  (complement)  of 
various  animals  differs  widely.  Guinea-pigs'  com- 
plement is  most  easily  fixed,  goats'  complement  being 
hardly  fixed  at  all.  The  complements  of  man,  horse, 
ox,  sheep,  and  rabbit  are  fixed  in  intermediate  and 
varying  degrees  to  those  mentioned  before.  In  per- 
forming these  experiments  two  different  amboceptors 
(both  specific  for  human  corpuscles)  produced  in 
rabbits  and  goats  were  used.  Kabbits'  complement 
possessed,  as  a  rule,  the  best  reactivating  property 
for  the  rabbits'  amboceptor,  but  the  complements  of 
sheep,  pig,  and  ox  seemed  far  inferior  or  often  devoid 
of  this  property.     Those  of  man,  goat,  and  horse 


SERUM  DIAGNOSIS  OF  SYPHILIS.  25 

are  weakly  reactivating  for  this  amboceptor.  The 
amboceptor  from  goats  could  be  reactivated  by  all  of 
the  above  sera  excepting  that  of  pigs,  although  goats' 
serum  was  most  effective  and  guinea-pigs'  somewhat 
less  so. 

We  have  also  seen  the  mode  of  detecting,  by  means 
of  the  precipitation  reaction,  an  unknown  antigen  or 
antibody  by  direct  observation.  ISTow  antigen  can  also 
be  detected,  by  indirect  observation,  through  the  em- 
ployment of  the  complement-fixation  reaction. 

To  illustrate  the  mechanism  of  the  Bordet-Gengou 
phenomena  I  introduce  (pp.  22,  23)  schematic  illus- 
trations based  upon  the  well-known  side-chain  theory 
of  Ehrlich.  Fig.  5  shows  three  different  combinations 
of  antigens  and  antibodies,  each  capable  of  grasping 
or  attracting  complement. 

Fig.  6  illustrates  the  deviation  of  complement  by 
one  combination  of  antigen  and  antibody  preventing 
the  complement  from  taking  part  in  another  reaction 
after  such  fixation. 

The  application  of  this  indirect  method  of  deter- 
mining the  presence  of  a  specific  reaction  between  an- 
tigen and  antibody  has  been  extensively  applied  to 
various  infectious  diseases  with  more  or  less  success. 
The  details  of  the  application  of  this  principle  to 
syphilis  will  be  developed  in  the  following  pages. 


IV. 


THE   APPLICATION   OF  THE   INDIRECT   METHOD   OF 

DETERMINING  ANTIBODIES  TO  THE 

DIAGNOSIS  OF  SYPHILIS. 

We  have  so  far  developed  the  fact  that  combina- 
tions of  antigen  and  antibody  which  do  not  require 
complements  to  complete  their  characteristic  reaction 
may  still  bind  complement  and  prevent  its  taking  part 
in  other  reactions.  We  have  also  noted  'that  the 
property  of  fixing  complement  may  be  exerted  by 
quantities  of  antigen  and  antibody  which  are  too  small 
to  give  rise  to  the  characteristic  reaction  of  such  a 
combination,  namely,  visible  precipitation.  From 
this  it  was  but  a  short  step  to  the  conception  that  there 
might  be  a  combination  of  antigen  and  antibody  for 
which  we  are  acquainted  with  no  characteristic  direct 
manifestation  but  which  could  still  exert  a  fixing 
effect  on  complement. 

From  clinical  studies  it  has  long  been  known  that 
syphilis  is  an  infectious  disease  which  in  running  its 
course  produces  a  specific  immunity.  That  an  im- 
munity is  developed  means,  presumably,  that  anti- 
bodies against  the  infectious  agent  are  produced  in 
the  subject  at  some  stage  of  the  process.  As  the  in- 
fectious agent  has  not  up  to  the  present  time  been 

26 


SERUM  DIAGNOSIS  OF  SYPHILIS.  27 

cultivated  or  by  other  means  separated  in  any  quantity 
from  the  diseased  tissues,  it  is  impossible  to  deter- 
mine the  presence  of  antibodies  by  the  direct  obser- 
vation of  such  a  reaction  as  bacteriolysis  or  aggluti- 
nation. Moreover,  even  though  we  accept  the  Trepo- 
nema (Sjnrochceta)  pallidum  as  the  cause  of  the  dis- 
ease, the  detection  of  its  presence  is  not  to  be  relied 
upon  as  our  only  means  of  diagnosis.  In  the  late 
manifestations  of  the  disease,  at  a  time  when  it  is  still 
infectious  and  still  amenable  to  specific  treatment, 
the  treponemata  are  present  in  such  small  numbers  as 
to  be  most  difficult  of  detection.  In  exactly  these 
cases  a  measure  of  immunity  may  be  supposed  to  have 
developed  and  specific  antibodies  to  have  been  formed. 

The  idea,  then,  was  to  take  syphilitic  tissues  at  a 
stage  when  the  treponemata  were  most  numerous  and 
use  this  as  the  known  antigen.  Tests  against  this 
known  antigen  with  blood-serum  of  other  patients 
might,  it  could  be  hoped,  reveal  the  presence  of  anti- 
body. As  has  been  said,  the  direct  method  of  obser- 
vation had  nothing  to  offer  and  the  indirect  method 
was  tried  from  the  first. 

The  earliest  publication  on  this  indirect  method 
of  detecting  the  syphilitic  antibody  is  that  by  Wasser- 
mann,  Neisser  and  Bruck  on  May  10,  1906,  and  the 
next  article  is  that  by  Ladislaus  Detre  on  May  24 
of  the  same  year.    These  investigators  were  working 


28  SERUM  DIAGNOSIS  OF  SYPHILIS. 

on  the  same  subject  independently  during  the  same 
period  of  time  and  obtained  exactly  the  same  results. 

The  technic  employed  by  Wassermann,  Neisser 
and  Bruck,  on  the  one  hand,  and  by  Detre,  on  the 
other,  was  almost  identical  except  in  small  details  and 
will  be  stated  here  in  a  general  way. 

Extracts  of  syphilitic  tissues  in  the  active  stages 
of  the  disease  were  made  and  used  as  antigen.  Was- 
sermann, Neisser  and  Bruck  used  the  liver  of  a  con- 
genitally  syphilitic  foetus,  and  Detre  employed  chiefly 
condylomata  for  this  purpose.  Serum  of  known 
syphilitics,  inactivated  at  56°  C,  was  used  as  anti- 
body. To  this  combination  complement  was  added. 
So  far  there  was  no  visible  change.  If  after  a  short 
time  a  quantity  of  immune  hasmolytic  amboceptor  was 
added  to  the  mixture,  and  then  the  cells  for  which  this 
amboceptor  was  developed,  no  hjKmolysis  took  place. 
It  can  be  shown  by  suitable  experiments  that  the  fail- 
ure of  the  erythrocytes  to  dissolve  is  not  due  to  any 
change  in  the  cells.  Neither  is  it  due  to  interference 
with  the  amboceptor.  The  complement  has  been  pre- 
vented from  acting,  has  been  fixed,  or  deviated.  If 
in  place  of  the  blood-serum  of  a  known  syphilitic  there 
had  been  used  as  unknown  antibody  the  blood  of  a 
person  known  never  to  have  had  syphilis,  haemolysis 
would  occur,  because  the  complement  was  not  inter- 
fered with.    The  erythrocytes  used  by  Wassermann, 


SERUM  DIAGNOSIS  OF  SYPHILIS.  29 

Neisser  and  Bruck  were  those  of  the  sheep,  and  those 
used  by  Detre  were  from  the  horse.  The  ambocep- 
tors employed  were  of  course  an  antisheep  serum  in 
the  first  instance  and  an  antihorse  serum  in  the  sec- 
ond, both  being  derived  from  rabbits  immunized  with 
the  erythrocytes  of  the  corresponding  animals. 

In  the  beginning  it  was  supposed  that  the  antigen 
also  was  specific ;  that  is,  that  the  serum  of  the  syphi- 
litic patient  would  only  fix  complement  in  the  presence 
of  extracts  of  syphilitic  tissues.  By  experiments  of 
Landsteiner,  Miiller  and  Potzl;  Levaditi;  Weil  and 
Braun;  of  Meier;  and  also  of  the  writer,  it  has, 
however,  been  determined  that  when  such  a  serum 
is  combined  with  the  alcoholic  extracts  of  certain 
normal  organs  or  with  a  preparation  of  crude  tissue- 
lecithin  complement  is  also  fixed.  Because  they  are 
easier  to  obtain  such  nonspecific  extracts  are  now 
commonly  used  as  antigens  in  this  reaction.  When 
the  diseased  tissues  are  used  as  the  source  of  antigen 
the  liver  of  a  syphilitic  foetus  is  commonly  chosen. 

Summary. — It  has  been  found  on  the  basis  of 
thousands  of  comparative  tests  that  if  the  blood-serum 
of  a  patient  suffering  from  syphilis  be  mixed  in  the 
presence  of  complement  with  extracts  of  syphilitic 
livers,  with  alcoholic  extracts  of  certain  normal 
organs,  or  with  crude  tissue-lecithin  preparations,  the 
complement  will  be  fixed  and  prevented  from  taking 


30  SERUM  DIAGNOSIS   OF  SYPHILIS. 

part  in  a  subsequent  haemolytic  reaction.  Starting 
with  the  extracts  given  and  mixing  with  them,  in  the 
presence  of  complement,  serum  of  unknown  origin, 
if  complement  be  fixed  it  can  be  stated  with  assur- 
ance (with  certain  reservations  to  be  discussed  later) 
that  the  unknown  serum  was  derived  from  a  case  of 
syphilis. 

To  those  who  have  carefully  considered  the  quan- 
titative relationship  of  the  factors  in  a  simple  reaction 
with  a  hasmolytic  serum  as  presented  in  the  first 
chapters  of  this  book,  it  will  at  once  be  apparent  that 
in  a  test  in  which  the  haemolytic  reaction  is  to  be 
used  as  an  indicator  of  a  second  reaction  not  other- 
wise visible,  the  quantitative  values  of  all  the  factors 
must  be  accurately  determined  and  rigidly  adhered  to 
if  the  results  are  to  be  relied  upon.  The  following 
chapter  is  devoted  to  the  consideration  of  the  quan- 
titative relationship  of  these  factors  and  to  certain 
other  precautionary  measures  which  must  be  observed. 


V. 


QUANTITATIVE  RELATIONS  OF  THE  FACTORS  IN   THE 
SERUM  DIAGNOSIS  OF   SYPHILIS. 

It  will  be  recalled  that  in  the  complement-fixation 
reaction  there  enter  into  operation  five  distinct  and 
essential  factors.  Enumerated,  they  are:  syphilitic 
antigen,  syphilitic  antibody,  erythrocytes,  hemolytic 
amboceptor  and  complement.  Two  of  these  fac- 
tors are  antigens,  the  erythrocyte  and  the  organic 
extract;  two  of  them  are  antibodies,  the  hgemolytic 
amboceptor  and  the  syphilitic  antibody.  To  avoid 
confusion  in  the  discussion  of  the  reaction  it  has  been 
customary  to  group  these  factors  according  to  their 
functions  as  follows:  the  erythrocytes,  the  hsemolytic 
amboceptor,  and  the  complement  are  collectively  re- 
ferred to  as  the  hcemolytie  system.  Speaking  of  the 
first  two  factors  of  the  heemolytic  system  it  is  cus- 
tomary to  avoid  the  terms  antigen  and  antibody  and 
speak  of  erythrocytes  and  amboceptor.  When  in 
connection  with  the  test  we  refer  to  antigen  and  anti- 
body, we  mean  only  the  two  factors  outside  the  haem- 
olytic  system,  the  syphilitic  antigen  and  the  anti- 
body present  or  absent  in  the  serum  to  be  tested.  In 
principle  any  haemolytic  system  can  be  used  as  an 

31 


32  SERUM  DIAGNOSIS  OF  SYPHILIS. 

indicator  for  the  test,  provided  the  complement  used 
is  sensitive  to  fixation. 

Whatever  system  be  chosen  the  relation  of  am- 
boceptor, complement,  and  erythrocytes  as  outlined 
in  the  second  chapter  must  be  observed.  That  is, 
one  must  work  with  a  suspension  of  erythrocytes  of 
definite  value,  the  amboceptor  must  be  carefully  ti- 
trated with  respect  to  that  erythrocyte  suspension,  and 
with  respect  to  the  complement  employed.  Then 
definite  amounts  of  amboceptor  must  be  used,  and 
the  quantity  of  complement  to  be  employed  must  be 
constant,  and  so  adjusted  as  to  act  with  the  quantity 
of  amboceptor  and  erythrocyte  suspension  deter- 
mined upon.  If,  for  example,  we  use  in  the  hasmo- 
lyiic  system  one  amboceptor  unit,  at  least  one  comple- 
ment unit  must  be  combined  with  this  in  order  to 
obtain  complete  hsemolysis.  If  less  than  one  com- 
plement unit  were  added  to  the  antigen-antibody  mix- 
ture in  the  fixation  test,  then  haemolysis  would  cer- 
tainly be  incomplete  and  one  might  imagine  that 
complement  was  fixed  when  it  was  merely  deficient 
in  quantity,  from  the  beginning.  If  many  ambo- 
ceptor units  are  used  haemolysis  may  be  complete  in 
the  presence  of  much  less  than  one  unit  of  comple- 
ment. Theoretically  the  test  for  fixation  of  comple- 
ment might  be  made  much  more  delicate  by  uniting 
two  units  of  amboceptor  with  only  a  fraction  of  one 


SERUM  DIAGNOSIS  OF  SYPHILIS.  33 

unit  of  complement.  In  practice,  however,  there  are 
certain  extrinsic  factors  which  may  interfere  with  the 
action  of  small  amounts  of  complement,  and  it  is 
therefore  not  safe  to  use  minimal  quantities.  These 
factors  will  be  more  carefully  and  fully  considered 
later. 

A  more  common  quantitative  error  is  the  follow- 
ing: Suppose  that  the  antigen-antibody  combination 
in  any  given  test  is  capable  of  binding  one  and  one- 
half  units  of  complement.  The  one-half  unit  of 
complement  remaining,  two  units  being  used  in  the 
test,  would  be  incapable  of  completing  haemolysis 
with  the  amount  of  amboceptor  which  should  be 
added,  but  with  a  high  multiple  of  that  amount  could 
easily  do  so.  We  would  then  obscure,  by  an  excess 
of  amboceptor,  a  positive  reaction.  If,  on  the  other 
hand,  with  a  small  amount  of  syphilitic  antigen  and 
antibody  we  use  an  excessive  amount  of  complement, 
the  antigen-antibody  combination  may  bind  comple- 
ment up  to  its  capacity  and  still  leave  a  sufficient 
quantity  to  act  in  conjunction  with  the  amboceptor 
to  produce  haemolysis.  We  would  then  again  have  a 
false  negative  instead  of  a  positive  reaction. 

It  was  stated  that  certain  factors  not  directly  in- 
volved in  the  reaction  might  interfere  with  it  if  smaU 
amounts  of  complement  are  used.  There  are  at  times 
substances  in  the  extracts  other  than  the  specific  an- 


84  SERUM  DIAGNOSIS  OF  SYPHILIS. 

tigen  which  prevent  complement  from  acting.  They 
are  commonly  known  as  anticomplementary  sub- 
stances. Anticomplementary  substances  may  also 
exist  in  the  serum  to  be  tested.  In  order  to  guard 
against  error  from  this  source  when  making  the  test 
with  preparations  with  more  or  less  anticomplemen- 
tary action  it  is  necessary  to  add  antigen  alone,  and 
the  test  serum  alone,  to  the  hemolytic  system  in  some- 
what larger  quantity  than  they  are  to  be  used  com- 
bined in  the  test.  Human  serum  may  contain  natural 
hasmolytic  amboceptors  for  the  erythrocytes  in  use 
when  the  erythrocytes  belong  to  alien  bloods.  Thus 
if  we  add  one  unit  of  immune  amboceptor  to  a  mix- 
ture already  containing  six  units  of  natural  ambocep- 
tor, it  would  produce  exactly  the  same  discrepancy  in 
result  as  though  the  immune  amboceptor  were  added 
in  excess.  This  source  of  error  is  to  be  avoided  by 
choosing  a  hsemolytic  system  for  which  human  serum 
contains  no  natural  hsemolytic  amboceptors.  There 
may  also  be  hamolytic  substances  in  the  antigen 
preparations.  These  must  be  tested  for  each  time 
when  deahng  with  unknown  or  freshly  prepared 
extracts. 

Complements  of  different  species  of  animals 
behave  differently  toward  the  fixation  by  antigen- 
antibody  combinations.  Certain  species  of  animals 
contain  in  their  sera  complements  which  are  readily 


SERUM  DIAGNOSIS  OF  SYPHILIS.  35 

fixed,  while  in  other  groups  of  species  their  comple- 
ments are  quite  refractory  or  not  at  all  susceptible  to 
the  fixation.  The  writer,  with  Bronfenbrenner,  made 
an  extensive  series  of  comparative  study  of  various 
complements  and  has  arrived  at  the  conclusion  that 
the  complement  contained  in  guinea-pig's  serum  is 
the  best  for  the  fixation  tests. 

Coming  now  to  the  exact  quantities  to  be  used; 
the  determination  of  the  strength  of  amboceptor  and 
complement  has  been  described  in  detail  (see  Chapter 
II).  It  is  customary  to  use  a  slight  excess  of  each, 
usually  two  units.  This  makes  the  reaction  some- 
what less  delicate,  but  allows  a  margin  for  error  due 
to  anticomplementary  substances  and  in  the  long  nm 
makes  the  test  more  reliable.  The  amount  of  serum 
to  be  tested  for  its  antibody  content  must  be  large 
enough  to  bind  aU  complement  in  the  presence  of 
sufficient  antigen  if  the  serum  be  from  a  known 
syphilitic  case.  The  proper  quantity  of  the  serum 
to  he  used  depends,  therefore,  upon  the  quantity  of 
the  complement  used  in  the  hcemolytic  system.  It  is 
obviously  of  advantage  to  construct  a  system  in 
which  as  smaU  an  amount  as  possible  of  patient's 
serum  can  be  employed  without  diminishing  the 
delicacy  or  reliability  of  the  reaction. 


VI. 

VARIOUS  FORMS  OF  THE  COMPLEMENT-FIXATION 
TEST  AS  APPLIED  TO  THE  SERUM  DIAG- 
NOSIS OF  SYPHILIS. 

As  was  pointed  out  in  the  last  chapter,  it  is 
erroneous  to  think  that  almost  any  haemolytic  system 
can  be  used  to  test  the  binding  power  of  antigen-anti- 
body combination  for  complement.  On  the  contrary 
the  experimenter  must  be  aware  of  the  disturbing 
effect  that  results  from  the  use  of  a  hsemolytic  system 
in  which  alien  erythrocytes  naturally  susceptible  to 
the  hsemolytic  action  of  human  serum  are  employed. 
Here  the  amount  of  the  amboceptor  becomes  uncon- 
trollable and  extremely  variable,  for  the  reason  that 
the  amounts  of  the  natural  amboceptor  contained  in 
the  serum  to  be  examined  are  unknovni  and  variable. 
The  second  important  point  which  the  experimenter 
must  always  bear  in  mind  before  employing  a 
hemolytic  system  is  the  quality  of  complement  in 
regard  to  fixation  phenomenon.  As  was  stated  in 
the  last  chapter,  different  complements  vary  con- 
siderably in  their  activity  and  fixability.  The  selec- 
tion of  the  haemolytic  system  is,  therefore,  one  of  the 
most  important  problems  that  involve  the  comple- 

36 


SERUM  DIAGNOSIS  OF  SYPHILIS.  37 

ment-fixation  tests  and  demands  the  utmost  care  and 
consideration  on  the  part  of  the  experimenter. 

Unfortunately,  chiefly  owing  to  the  lack  of  exact 
experimental  knowledge  of  these  facts  in  the  earlier 
period  of  evolution  of  the  complement-fixation  tests, 
various  investigators  proposed  the  use  of  this  or  that 
heemolytic  system  as  long  as  they  could  produce  the 
phenomenon  of  haemolysis. 

When  one  proceeds  to  consider  one  h^emolytic 
system  after  another  from  a  quantitative  view-point 
he  will  not  have  much  difficulty  in  judging  the  real 
merit  of  each  method.  Some  of  them  are  such  as  to 
permit  a  qualitative  determination  of  the  reaction, 
while  others  are  so  crude  that  even  this  much  can  not 
be  accomplished.  If  we  are  to  advance  in  the  serologi- 
cal field  it  is  our  duty  to  advocate  that  system  in  which 
every  factor  entering  into  the  fixation  tests  is  under 
a  perfect  quantitative  control  by  the  experimenter. 
At  least  twelve  different  systems  have  been  proposed 
up  to  the  present  time.  These  systems  can  be  divided 
into  two  groups  according  to  whether  foreign  or 
human  corpuscles  are  used  as  the  hsemoljrtic  indicator. 
A  brief  critical  review  of  these  will  perhaps  make 
clearer  the  principles  of  the  test. 

Wassermann,  Neisser  and  Bruck  use  sheep  blood- 
corpuscles,  an  immune  heemolytic  amboceptor  made 
by  immunizing  a  rabbit  with  sheep's  erythrocytes,  and 


88  SERUM  DIAGNOSIS  OF  SYPHILIS. 

guinea-pig  complement.  In  making  the  test  the 
syphilitic  serum  is  inactivated.  The  quantity  used 
is  0.1  c.c.  and  0.2  c.c.  for  each  specimen  of  serum.  Two 
units  of  the  amboceptor  and  0.1  c.c.  of  guinea-pig's 
complement  are  used  against  1.0  c.c.  of  a  5  per  cent, 
suspension  of  the  washed  sheep-corpuscles.  The  re- 
sultant volume  of  the  whole  mixture  is  brought  up 
uniformly  to  5  c.c.  There  is  but  one  large  factor  of 
error  and  that  operates  as  follows :  There  is  in  human 
serum  a  variable  amount  of  natural  antisheep  ambo- 
ceptor. In  the  cases  in  which  such  an  amboceptor  is 
present  in  appreciable  quantity  it  serves  to  increase 
the  total  effective  amboceptor  in  the  mixture.  This, 
according  to  the  relationship  existing  between  the 
amount  of  amboceptor  and  complement  respectively 
required  for  complete  haemolysis,  tends  always  to 
make  haemolysis  complete  even  when  antigen-antibody 
has  fixed  a  considerable  amount  of  the  complement 
(Figs.  2,  3,  and  4,  and  references  to  previous  chap- 
ters). It  is  also  possible  that  the  complement  which 
has  been  completely  fixed  by  a  moderate  amount  of 
antigen-antibody  combination  may  become  once  more 
detached  if  the  amount  of  the  haemolytic  amboceptor 
introduced  be  very  large,  and  produce  complete 
haemolysis.  As  the  occurrence  of  haemolysis  means  a 
negative  reaction,  or  absence  of  syphilitic  antibody, 
the  error  in  this  case  is  always  in  the  direction  of 


SERUM  DIAGNOSIS  OF  SYPHILIS.  39 

throwing  sera  with  smaller  amounts  of  syphiHtic  anti- 
body into  the  negative  class.  If  an  error  in  diagnosis 
is  inevitable  it  is  of  course  safer  to  have  it  in  this 
direction ;  but,  as  will  be  pointed  out  later,  this  source 
of  error  can  be  avoided  by  a  change  in  the  haemolytic 
system. 

Bauer  in  his  test  relies  entirely  for  amboceptor 
upon  the  natural  antisheep  amboceptor  in  human 
serum,  which,  as  has  been  pointed  out,  is  a  source  of 
error  in  Wassermann's  system.  This  is  merely  a 
makeshift  and  does  not  eliminate  the  error,  because 
amboceptor  is  not  always  naturally  present  and  when 
present  varies  greatly  in  quantity.  For  this  reason 
the  test  is  unreliable,  because  of  oversensitiveness, 
since,  as  pointed  out,  amboceptor  may  not  be  pres- 
ent at  all  or  only,  as  may  happen,  in  a  fraction  of 
one  unit. 

Hecht  relies  not  only  on  the  natural  antisheep 
amboceptor  of  human  serum  but  also  on  human  com- 
plement. It  will  be  seen  at  once  that  if  this  were  a 
system  whose  factors  were  regular  in  quantity,  it 
would  be  much  simpler  in  practice  than  Wassermann's 
system.  All  that  would  be  necessary  would  be  to  add 
antigen  to  the  patient's  own  serum,  which  would  con- 
tain complement  and  antibody  to  be  detected,  and 
amboceptor.  After  a  period  of  incubation  sheep 
erythrocytes  would  be  added  and  the  test  read  "  posi- 


40  SERUM  DIAGNOSIS  OF  SYPHILIS. 

tive  "  or  "  negative  "  after  another  period  of  incuba- 
tion. However,  not  only  is  the  amboceptor  a  factor 
varying  from  zero  to  ten  units  or  more,  but  human 
complement  is  far  less  regular  in  activity  and  fixabil- 
ity  than  is  guinea-pig's  complement.  Further,  the  test 
would  have  to  be  done  with  fresh  serum  or  the  comple- 
ment would  surely  be  reduced  or  totally  lost  by 
spontaneous  deterioration.  There  is  not  in  this 
system  a  direct  way  of  testing  the  anticomplementary 
action  of  antigen  alone. 

M.  Stern  proposed  a  system  in  which  there  were 
added  a  few  units  of  immune  antisheep  amboceptor 
to  the  fresh  serum  to  be  tested,  utilizing  the  comple- 
ment of  the  patient's  serum.  This  still  retains  all  the 
defects  inherent  in  the  use  of  unknown  and  often  ex- 
cessive amount  of  the  h^emolytic  amboceptor,  and 
makes  it  impossible  to  test  a  specimen  that  has  been 
kept  a  few  days  after  collection.^ 

Detre  and  Brezovsky  used  horse  corpuscles,  an 
immune  antihorse  hemolytic  amboceptor  derived 
from  a  rabbit  injected  with  these  erythrocytes,  and 
rabbit's    complement.      As   human    serum    contains 

*  According  to  my  recent  investigations  certain  proteins,  such  as 
pepton,  albumoses,  nucleoproteins,  and  certain  peptids  can  often  produce 
complement  fixation  when  mixed  with  unheated  human  serum,  closely 
resembling  specific  lixation.  For  this  reason  no  active  serum  should 
be  used  for  the  test  with  aqueous  or  even  alcoholic  extracts  of  liver, 
especially  of  macerated  organs.  Pure  lipoids  free  from  above  mentioned 
substances  do  not  give  this  false  fixation  with  active  serum  (Noguchi). 


SERUM  DIAGNOSIS  OF  SYPHILIS.  41 

natural  antihorse  amboceptor  to  about  the  same  de- 
gree and  frequency  as  antisheep  amboceptor,  this 
system  is  no  more  reliable  than  Wassermann's  system. 
Further,  the  reagents  are  difficult  to  procure. 

Boas  advocated  a  system  similar  to  the  Wasser- 
mann  system,  using  an  antigoat  amboceptor  produced 
in  rabbits.    Later  this  is  abandoned  by  him. 

Browning  used  an  anti-ox  amboceptor  produced 
in  rabbits,  and  claims  that  human  serum  does  not  con- 
tain a  disturbing  excess  of  anti-ox  amboceptor. 

Tschernogubow  proposed  a  system  in  which  the 
natural  amboceptor  and  complement  of  human  serum 
are  utilized  against  guinea-pig  corpuscles. 

Foix  used  a  system  in  which  the  natural  ambo- 
ceptor and  complement  of  human  serum  are  utilized 
against  the  corpuscles  of  rabbit.  These  two  last  men- 
tioned systems  neglect  entirely  the  quantitative  phases 
of  haemolysis. 

Kaliski  has  introduced  a  system  in  which  active 
patient's  serum  is  used  with  the  addition  of  guinea- 
pig  complement — thus  disregarding  the  native  com- 
plement of  the  human  serum — and  antisheep  hsemo- 
lytic  system.  This  ought  not  to  be  classed  with  the 
system  of  Stern,  for  the  reason  that  Kaliski  uses  a 
much  smaller  proportion  of  patient's  serum  and  re- 
duces the  disturbing  eiFect  of  the  natural  antisheep 
amboceptor  and  native  complement  contained  in  the 


42  SERUM  DIAGNOSIS  OF  SYPHILIS. 

specimen  and  that  guinea-pig  complement  is  being 
employed. 

These  nine  different  systems  may  be  considered 
as  of  one  general  order,  having  in  common  the  use 
of  erythrocytes  of  animals  for  which  human  serum 
contains  an  unknown  and  irregular  quantity  of 
natural  h^emolytic  amboceptors.  When  subdivided 
according  to  the  varieties  of  complements  employed 
it  will  be  seen  that  the  systems  of  Wassermann,  Bauer, 
Boas,  and  Browning  use  guinea-pig  complement; 
those  of  Hecht,  Stern,  Tschernogubow  and  Foix  the 
native  complement  of  patients'  serum;  Detre  that  of 
rabbit;  and  finally  Kaliski  a  mixture  of  human  and 
guinea-pig  complements.  In  none  of  these  systems 
can  the  natural  amboceptors  be  removed  or  put  under 
quantitative  regulation.  In  fact,  some  systems 
utilize  them  to  produce  haemolysis,  while  others  simply 
disregard  their  presence  and  add  to  every  instance  a 
certain  amount  of  immune  amboceptors  to  provide  for 
occasional  absence  or  deficiency  of  natural  ambo- 
ceptors. 

In  the  following  we  now  take  up  the  systems  in 
which  human  erythrocytes  are  employed. 

Tschernogubow,  in  an  article  published  several 
months  before  his  second  system,  discussed  with  the 
foregoing  group,  also  proposed  a  system,  which  he 
has  since  abandoned,  with  quite  a  different  set  of 


SERUM  DIAGNOSIS  OF  SYPHILIS.  43 

factors.  In  general,  it  has  some  resemblance  to 
the  system  put  forward  at  about  the  same  time  by 
the  author.  Both  systems  use  human  erythrocytes 
and  antihuman  hsemolytic  amboceptor,  but  the  source 
of  complement  and  the  manner  of  conducting  the 
test  are  altogether  different.  Tschernogubow  collects 
the  patient's  blood  (not  serum)  in  saline  solution 
in  such  dilution  that  clotting  is  temporarily  prevented. 
This  suspension  when  fresh  contains  some  comple- 
ment, erythrocytes,  and,  if  present,  the  syphilitic  anti- 
body. When  the  antigen  is  added  the  antibody 
unites  with  it  and  the  combination  fixes  comple- 
ment. The  antihuman  amboceptor  is  added  later, 
when  haemolysis  occurs  in  case  the  complement  has 
not  been  fixed.  This  system  shows  four  sources  of 
error.  ^  The  amount  of  complement  in  human  serum 
is  irregular  and  its  activity  is  weak  in  relation  to 
antihuman  heemolytic  amboceptor.  The  complement 
and  erythrocytes  deteriorate  rather  rapidly,  hence  no 
examination  can  be  made  of  old  specimens.  The  com- 
plement cannot  be  put  in  contact  with  antigen  alone 
and  consequently  it  is  impossible  to  decide  if  the 
antigen  is  or  is  not  inherently  anticomplementary  by 

*  A  fifth  source  of  error  has  since  been  discovered  by  me,  that  is, 
the  use  of  active  serum  in  combination  with  aqueous  extracts  of  liver 
renders  the  test  nonspecific.  This  objection  applies  to  any  other  system 
using  active  serum  and  aqueous  or  even  alcoholic  extracts  (unfrac- 
tionated)   of  macerated  livers. 


44  SERUM  DIAGNOSIS  OF  SYPHILIS. 

a  direct  test  (Chapter  V).  Strangely  enough, 
Tsehernogubow  did  not  state  the  source  of  his  anti- 
human  amboceptor.  He  does  say  that  0.25  c.c.  was 
added  to  each  tube.  This  is  nearly  125  times  the 
amount  of  amboceptor  I  use  in  combination  with 
guinea-pig  complement.  It  is  probable  that  in  this 
way  he  overcomes,  in  a  measure,  the  variations  in  the 
amount  of  human  complement  present.  Still,  the 
uncertainty  in  this  respect,  the  impossibility  of 
separating  the  factors  in  the  system  for  control,  and, 
lastly,  the  necessity  of  making  the  test  very  soon  after 
the  collection  of  the  blood  in  order  that  complement 
may  be  active  and  the  erythrocytes  intact,  leave 
abundant  room  for  improvement  and  render  the 
system  as  outlined  too  unreliable  for  general  use. 

After  considerable  experience  with  the  Wasser- 
mann  test  in  its  original  form,  using  both  the  aqueous 
and  the  alcohoUc  extracts  as  antigen,  I  became  con- 
vinced of  its  value  in  the  diagnosis  of  syphilitic  and 
parasyphilitic  conditions.  I  felt,  however,  that  if  the 
reaction  was  to  come  into  general  use  it  would  have 
to  be  greatly  simplified.  In  attempting  such  a  simpli- 
fication it  was  essential  that  nothing  making  for  ac- 
curacy be  sacrificed.  It  was  thought  that  it  would  be 
of  immense  advantage  to  eliminate,  if  possible,  the 
error  due  to  the  irregular  presence  of  natural  antisheep 
amboceptor  in  human  serum.    The  directions  in  which 


SERUM  DIAGNOSIS  OF  SYPHILIS.  45 

simplification  was  most  needed  will  be  briefly  alluded 
to.  The  Wassermann  test  demanded  the  use  of 
fresh  washed  sheep's  corpuscles  each  time  a  test  was 
to  be  made.  Persons  far  removed  from  a  large 
abattoir  would  have  difficulty  in  obtaining  sheep's 
blood.  Washing  the  corpuscles  was  essential,  and 
required  a  good  centrifuge.  The  serum  must  be  in- 
activated, demanding  care  and  a  water-bath.  The 
measuring  and  graduation  of  dosage  of  the  liquid 
preparations  require  a  full  equipment  of  laboratory 
glassware.  On  the  whole,  the  labor  was  so  great  that 
even  in  a  fully  equipped  laboratory  a  man  who  pro- 
posed to  carry  out  the  test  as  a  routine  procedure 
could  do  little  else;  and  outside  such  a  laboratory  the 
performance  of  the  test  was  not  to  be  thought  of.  In 
the  method  which  eliminates  these  difficulties,  a  dilute 
suspension  of  human  erythrocytes  is  used.  This  is 
readily  prepared  at  any  time  by  pricking  the  finger 
of  the  patient  and  allowing  the  blood  to  drop  into 
physiological  salt  solution.  It  has  been  found  possi- 
ble to  prepare  antigen,  antihuman  haemolytic  ambo- 
ceptor, and  guinea-pig  complement,  in  the  form  of 
reagent  papers,  which  remain  stable  for  a  long  time  if 
kept  perfectly  dry  and  air-tight.  The  factors,  it  will 
be  noted,  are  each  separate  and  distinct,  more  so 
than  in  the  Wassermann  system.  By  using  an  anti- 
human  haemolytic  system  the  variable  antisheep  ambo- 


46 


SERUM  DIAGNOSIS  OF  SYPHILIS. 


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48  SERUM  DIAGNOSIS  OF  SYPHILIS. 

ceptor  of  human  serum  is  eliminated  as  a  disturbing 
factor.  The  advantage,  in  point  of  regularity  and 
uniformity  gained  by  the  use  of  guinea-pig  comple- 
ment, is  retained  without  any  sacrifice  in  accurac}'-. 
And,  further,  the  test  can  be  put  within  reach  of  any 
physician  who  is  engaged  in  doing  laboratory 
work,  after  some  training  in  this  phase  of  haemolytic 
work.  Perhaps  of  more  real  advantage  to  the  pro- 
fession is  the  circumstance  that  blood-serum  collected 
by  the  physician  may  be  sent  to  a  public  laboratory 
and  there  examined.  The  writer's  system  renders 
the  test  so  simple  and  easy  that  the  method  may  well 
be  placed  on  the  Hst  of  regular  examinations  made  by 
most  hospital  laboratories.  The  details  of  the  method, 
with  directions  for  preparing  the  reagents  for  use  and 
the  detailed  directions  for  carrying  out  the  test  and 
interpreting  the  results,  I  have  brought  together  in 
another  chapter  of  this  book.  The  matter  presented 
in  regard  to  the  different  forms  of  the  complement- 
fixation  test  is  summarized  in  Table  1,  on  pages  46 
and  47. 

Von  Dungern  has  recently  introduced  a  system  somewhat  similar 
to  that  of  the  writer.  Like  Tschernogubow,  however,  he  uses  the 
patient's  blood  in  active  state  only,  for  the  reason  that  inactivation 
is  not  applicable  on  account  of  his  utilizing  the  erythrocytes  of  the 
same  blood.  Unlike  the  system  of  Tschernogubow  the  blood  is  defib- 
rinated  before  use,  but  the  native  complement  contained  in  it  is  not 
used  as  the  complement  constituting  the  haemolytic  system.  He 
advises  the  use  of  guinea-pig's  serum  in  dried  paper  form.    The  amount 


SERUM  DIAGNOSIS  OF  SYPHILIS.  49 

of  the  defibrinated  blood  used  for  the  test  is  0.1  c.c,  a  quantity  rather 
large,  and  the  presence  of  the  native  complement  there,  although  dis- 
regarded by  this  author,  can  hardly  be  left  out  of  consideration.  More- 
over, he  uses  an  entire  alcoholic  extract  (without  fractionation)  as 
antigen,  thus  introducing  a  possibility  of  obtaining  occasional  non- 
specific proteotropic  fixation  with  active  human  sera.  The  amboceptor 
used  by  von  Dungern  he  derives  from  the  immunized  goat.  This  adds 
another  source  of  uncertainty  of  the  result,  because  the  writer  had 
previously  found  that  this  amboceptor  gives  a  much  weaker  fixation 
reaction  than  that  derived  from  the  rabbit.  Von  Dungern  omits 
the  controls  with  a  positive  syphilitic  serum  and  also  with  the  usual 
haemolytic  system.  The  writer  believes  that  this  omission  is  to  be 
regretted.  Further  disadvantages  inherent  to  this  system  are  that 
there  is  no  direct  way  of  determining  the  anticomplementary  action 
of  antigen;  that  no  specimen  of  blood  older  than  a  few  days  can  be 
examined  on  accoxmt  of  the  weakening  of  the  erythrocytes  by  standing 
and  that  cerebrospinal  fluid  does  not  contain  erythrocytes,  hence  needs 
addition  of  the  latter  from  other  source  (blood).  It  will  be  seen  that 
this  system  is  not  quantitatively  constructed  and  offers  many  diffi- 
culties in  obtaining  reliable  results.  The  writer  warns  the  reader  not 
to  confuse  his  system  with  those  of  Tschernogubow  and  of  von 
Dungern,  as  seems  to  have  been  unjustly  done  by  a  few  German  authors. 


VII. 

A  SYSTEM  OF  SERUM  DIAGNOSIS  OF  SYPHILIS,  RECOM- 
MENDED BY  THE  AUTHOR. 

In  the  following  pages  it  is  my  purpose  to  present 
as  briefly  as  consistent  with  the  necessary  detail  the 
method  of  making  a  diagnosis  of  syphihs  by  serum 
reaction  as  it  has  been  developed  in  my  hands.  The 
presentation  will,  it  is  hoped,  be  of  interest  and  ser- 
vice to  two  distinct  sets  of  investigators,  viz.,  the  prac- 
tising physician  who  is  so  situated  that  he  must  make 
his  own  clinical  laboratory  tests  or  pass  them  by  en- 
tirely, and  those  laboratory  workers  who  are  con- 
cerned either  in  making  laboratory  diagnostic  tests 
for  others,  or  are  engaged  in  supplying  laboratory  re- 
agents in  convenient  and  stable  form  for  others  to 
use.  The  writer  must  make  it  clear  that  it  is  not  the 
adjective  "  practising  "  or  "  laboratory  "  that  qualifies 
a  physician  to  perform  the  serum  diagnosis.  One 
must  not  overlook  the  fact  that  among  the  practising 
physicians  there  are  as  many  competent  laboratory 
workers  as  there  are  unskilled  ones  among  the  labora- 
tory workers.  A  physician  who  has  had  regxilar  clini- 
cal laboratory  training  early  in  his  professional  career 
and  is  now  out  for  practice  is  certainly  qualified  to 
take  up  this  special  technic  of  diagnosis,  as  long  as  he 

50 


SERUM  DIAGNOSIS  OF  SYPHILIS.  51 

is  willing  to  divide  his  time  between  his  private  labora- 
tory work  and  practice.  No  matter  in  what  labora- 
tory, none  is  qualified  to  make  a  responsible  diagnosis, 
unless  the  performer  is  sufficiently  skilful  in  serologi- 
cal work.  In  reality  the  writer  knows  many  practis- 
ing physicians  who  after  acquiring  the  technic 
correctly  now  make  the  tests  with  the  reagents  secured 
from  others,  not  only  for  themselves,  but  also  for 
others,  while  a  great  many  bacteriologists  and  path- 
ologists will  be  at  a  loss  if  they  are  requested  to  make 
the  tests  before  they  are  trained  in  this  special  work. 

It  is  a  regrettable  phenomenon,  indeed,  to  see  that 
certain  practising  physicians  who  learned  to  do  this 
special  work,  present  themselves  now  as  competent 
workers  and  raise  loud  protests  against  others  who 
intend  to  take  up  the  test  similarly.  Another  group  of 
workers  protest  still  louder  against  the  diffusion  of 
this  method  and  these  are  found  among  the  regular 
laboratory  workers.  This  attitude  is  certainly  unjus- 
tified in  view  of  the  great  value  of  the  test.  We  all 
must  hope  that  the  test  will  diffuse  as  far  as  it  can 
among  physicians. 

The  real  duty  of  laboratory  workers  to-day  is  to 
improve  and  simplify  the  original  method  to  such  an 
extent  that  it  can  be  trusted  to  those  who,  while  unable 
to  prepare  the  reagents  themselves,  can  be  so 
instructed  as  to  be  able  to  carry  out  the  test  reliably. 


52  SERUM  DIAGNOSIS  OF  SYPHILIS. 

The  chief  cause  why  the  original  method  of  Wasser- 
mann  was  so  difficult  to  carry  out  is  because  in  that 
system  the  reagents  are  so  unstable  and  associated 
with  many  other  undesirable  side  actions  that  these 
must  be  used  soon  after  being  prepared.  This  cir- 
cumstance demands  that  the  performer  must  know 
how  to  prepare  the  reagents  and  to  make  the  test  all 
by  himself.  These  difficulties  are  inherent  to  most 
of  the  modifications  already  described.  On  the  other 
hand,  if  we  develop  the  method  in  such  a  manner  that 
the  preparation  of  the  most  important  and  difficult 
reagents  is  done  by  competent  laboratory  workers  in 
a  regular  biological  laboratory  and  distributed  among 
those  who  understand  how  to  use  them,  it  can  not 
bring  about  any  abuse  of  this  test.  The  writer  believes 
that  his  system  offers  this  advantage,  and  also  that 
the  reagents  as  recommended  in  his  system  remain 
unchanged  for  a  very  long  period.  The  performer 
of  the  test  should,  however,  know  how  to  determine 
the  reliability  of  the  reagents  he  secures  from  others. 
The  presentation  is  accordingly  made  in  two 
distinct  sections: 

A.  A  presentation  of  the  method  of  work  to  be 
followed  by  those  who  have  obtained  their  reagents 
from  others  and  who  must  make  their  own  tests. 

B.  A  description  of  the  method  of  preparing  the 
reagents,    standardizing   them,   preserving  them   in 


SERUM  DIAGNOSIS  OF  SYPHILIS.  53 

stable    form,    and    using    them    in    fully    equipped 
laboratories. 

The  interpretation  of  results  will  be  the  same  in 
both  instances  and  to  avoid  repetition  the  description 
of  this  subject  is  confined  to  Section  A. 

Section  A. 

METHOD   OF   WORK   TO   BE   FOLLOWED   BY   THOSE   WHO 

HAVE   OBTAINED    THEIR   REAGENTS   FROM    OTHERS 

AND  WHO  MUST  MAKE  THEIR  OWN  TESTS. 

For  making  the  test,  aside  from  the  reagents,  the 
following  special  apparatus  will  be  needed:  Several 
pipettes  of  1  c.c.  capacity  graduated  to  0.1  c.c. ;  two 
10  c.c.  pipettes  graduated  to  0.1  c.c;  several  1  c.c. 
pipettes  graduated  to  0.01  c.c;  a  number  of  small 
test-tubes,  the  best  dimension  being  10  x  1  cm.  (two 
tubes  mil  be  required  for  each  test  and  four  tubes 
for  controls  in  each  series  of  tests;  the  total  number 
needed  will,  of  course,  be  dictated  by  the  amount 
of  work  to  be  done)  ;  a  number  of  larger  test-tubes 
or  very  small  flasks  for  mixing  the  blood  suspension ; 
a  number  of  larger  flasks  or  bottles  as  containers  of 
physiological  salt  solution;  a  number  of  pieces  of  thin 
glass  tubing  about  4  mm.  in  bore  for  making  capillary 
pipettes.  A  few  test-tube  racks  with  two  parallel 
rows  of  holes  are  necessary. 

In  handling  the  preparations  and  glassware 
absolute  asepsis  is  not  required,  but  it  is  well  to  be 


54  SERUM  DIAGNOSIS  OF  SYPHILIS. 

reasonably  clean,  bacteriologically  speaking.  Physi- 
ological salt  solution  (0.9  per  cent.)  should  have  been 
boiled  before  use  and  then  cooled.  Glassware  should 
be  thoroughly  rinsed  with  boiling  water  and  al- 
lowed to  dry  without  wiping.  Chemical  cleanliness 
is  essential.  The  erythrocyte  is  a  delicate  cell  which  is 
most  easily  destroyed  or  altered  by  many  chemical 
substances  in  small  amounts.  Those  which  are  most 
apt  to  be  encountered  ordinarily  are  soaps,  weak 
solutions  of  mineral  acids  and  caustic  or  carbonated 
alkalies,  and  bichloride  of  mercury.  Test-tubes  which 
have  been  in  contact  with  any  of  these  substances  must 
be  thoroughly  washed  and  rinsed  in  clear  running 
water,  finally  being  boiled  in  pure  water  and  dried 
previous  to  use  in  the  test.  They  should  be  heated 
to  200°  C.  in  a  dry  air  sterilizer  before  use. 

Direct  preparation  for  making  the  test  includes 
the  following  procedures: 

COLLECTION    OF    SERUM    TO    BE    TESTED,* 

Only  about  1  c.c.  of  the  patient's  blood  is  needed. 
The  writer  has  found  it  very  convenient  to  obtain  it  by 
puncturing  the  ventral  side  of  the  last  joint  of  the 
middle  finger  with  a  sterile  Hagedorn  needle.  Before 
puncturing  compress  the  finger  tightly  by  squeezing 

*  Forbid  the  patient  to  take  any  alcoholics  48  hours  previous  to  the 
time  of  collecting  the  blood.  Craig  and  Nichols  were  the  first  to  find 
out  that  the  active  principle  of  the  Wassermann  reaction  disappears 
temporarily  when  alcohol  is  given  to  the  patient  or  syphilitic  animals. 


SERUM  DIAGNOSIS  OF  SYPHILIS. 


55 


it  in  such  a  manner  as  to  drive  the  blood  towards 
the  extremity  of  the  finger.  In  small  children 
the  lobe  of  the  ear  should  be  punctured  for 
greater  convenience.  The  blood  will  come  out  in 
drops.  For  collecting  the  blood  Wright's  capsules 
are  best  suited.     In  order  to  get  enough  blood  it 


Hejoested  iqueezincjto  ctriuelhe 
blood  foujarU  ihe  e^fretn'iTl/  of -the 
finger  is  vacesi&r^ 

\ 


(JOrlcjhth  capsule 


Tnar.nef  afcollectinfjih?  bhosl 


CfCer  Ihepiincta  re 
Fig.  7.— Showing  the  manner  of  collecting  the  blood  with  a  Wright's  capsule. 

is  usually  necessary  to  massage  the  finger  towards  the 
point  repeatedly.  One  puncture  usually  suffices. 
After  sufficient  blood  is  collected  seal  the  straight, 
empty,  capillary  end  with  a  flame,  then  cool  that  end. 
When  cool,  shake  the  capsule  to  drive  the  blood  from 
the  bent  end  to  the  straight  sealed  end ;  then  the  bent 
end  is  sealed  with  a  flame  in  turn.  By  this  means  the 
capsule  can  be  sealed  without  applying  the  heat  to  the 


56  SERUM  DIAGNOSIS  OF  SYPHILIS. 

blood  (Figs.  8  and  9).  Wright's  capsules  may  be 
made  by  drawing  out  ordinary  thin  glass  tubing  in  the 
flame  of  a  good-sized  alcohol  lamp,  or,  better,  a 
Bunsen  burner. 

The  blood  clot  and  the  serum  separate  in  a  few 
hours  at  room  temperature.  If  the  test  is  not  to  be 
made  within  two  or  three  days  the  serum  should  be 
drawn  ofl*  with  a  capillary  pipette  for  storage.  If 
left  in  contact  with  the  clot  it  will  finally  become 
tinged  with  hajmoglobin  and  this  will  somewhat  in- 
terfere with  the  accurate  reading  of  the  test  subse- 
quently. 

PREPARATION  OF  THE  CORPUSCLE  SUSPENSION. 

The  suspension  can  be  prepared  with  the  blood  of 
the  patient  being  examined.  When  several  or  more 
patients  are  being  examined  on  the  same  day  select  one 
of  them  to  draw  enough  blood  for  preparing  sufficient 
amount  of  suspension  for  all  the  rest  of  cases.  The 
standard  amount  of  corpuscle  suspension  for  my 
system  is  1  c.c,  of  a  1  per  cent,  or  0.1  c.c.  of  a  10  per 
cent,  for  each  tube.  Thus  if  we  draw  1  c.c.  of  blood 
from  any  of  the  patients  it  will  give  enough  corpuscles 
to  distribute  in  100  test-tubes.  The  most  important 
condition  that  must  be  strictly  observed  in  utilizing 
the  corpuscles  of  other  patients  is  the  complete 
removal  of  serum  from  the  suspension.  This  is  easily 
accomplished  by  washing  the  blood  by  means  of  cen- 
trifugalization.     I  will  describe  below  two  different 


.2-H 


r,    Q    a 
^   Rl   >■ 

5  S 


SERUM  DIAGNOSIS  OF  SYPHILIS.  67 

ways  of  making  suspension.  Take  a  graduated  centri- 
fuge tube  (capacity  10  c.c.)  and  fill  it  with  sodium  cit- 
rate solution  ^  up  to  9  c.c.  Allow  the  blood  of  patient 
to  drop  until  it  fills  up  to  10  c.c.  (9  c.c.  of  citrate 
solution  +  1  c.c.  blood).  The  blood  is  mixed  well 
with  the  citrate  solution  and  then  centrifugalized. 
Pour  off  the  supernatant  fluid  (containing  the 
serum)  and  fill  up  to  10  c.c.  with  a  fresh  lot  of  salt 
solution.  Stir  up  and  centrifugalize  again.  The 
supernatant  fluid  is  once  more  decanted  off.  The  de- 
posit (corpuscles  free  from  serum)  is  now  resuspend- 
ed  either  in  100  c.c.  of  salt  solution  (making  a  1  per 
cent,  suspension)  or  in  10  c.c.  (making  a  10  per  cent, 
suspension) .  In  the  test  one  uses  either  1  c.c.  of  the 
one  per  cent,  or  0.1  c.c.  of  the  ten  per  cent,  suspension 
for  each  tube  according  to  whether  the  experimenter 
prefers  the  first  or  the  second  procedure  which  will  be 
mentioned  later. 

When  one  has  defibrinated  blood  the  suspension 
may  be  similarly  made  after  removing  the  serum  by 
washing  with  a  large  amount  of  salt  solution.  Here 
one  cubic  centimetre  of  the  defibrinated  blood  will 
also  give  enough  suspension  for  100  tubes. 

The  use  of  the  one  per  cent,  corpuscle  suspension 
necessarily  introduces  the  maximum  volume  of  fluid 
for  each  tube  and  no  further  addition  of  salt  solution 
is  required.     On  the  other  hand,  the  employment  of 

^  This  is  prepared  by  adding  20  grammes  of  sodium  citrate  to  1000 
c.c.  of  0.9  per  cent,  salt  solution. 


68  SERUM  DIAGNOSIS  OF  SYPHILIS. 

the  ten  per  cent,  suspension  introduces  into  each  tube 
only  0.1  c.c.  of  fluid  and  demands  the  addition  of 
further  0.9  c.c.  of  salt  solution  to  come  up  to  the 
standard  volume  1  c.c.  for  each  tube.  The  conse- 
quence of  using  the  one  per  cent,  or  ten  per  cent, 
suspension  is  as  follows:  As  various  other  factors, 
such  as  complement,  antigen  and  patient's  serum,  are 
contained  in  very  small  quantities  of  fluid,  they  can 
not  react  with  each  other,  unless  they  are  put  together 
in  more  fluid.  In  order  to  accomplish  this  one  has 
to  make  up  the  total  volume  of  each  tube,  at  least,  1 
c.c.  This  can  be  done  either  by  adding  1  c.c.  of 
simple  salt  solution  or  1  c.c.  of  the  one  per  cent, 
corpuscle  suspension.  Thus  one  will  quickly  see  that 
in  case  of  using  the  one  per  cent,  corpuscle  suspension 
the  addition  of  corpuscles  is  necessarily  done  at  the 
very  beginning  of  making  the  test.  On  the  other  hand, 
when  the  ten  per  cent,  suspension  is  used  the  addi- 
tion of  corpuscles  may  be  made  afterwards,  as  one 
can  use  salt  solution  without  corpuscles  at  the  start. 
The  method  in  which  1  c.c.  of  the  one  per  cent, 
suspension  is  used  from  the  beginning  will  be 
called  the  first  and  that  in  which  0.1  c.c.  of  the  ten 
per  cent,  suspension  is  added  later  the  second  pro- 
cedure. The  advantage  of  the  second  procedure 
over  the  first  is  that  one  can  add  amboceptor  from 
the  beginning  (as  there  are  no  corpuscles  yet). 


SERUM  DIAGNOSIS  OF  SYPHILIS.  69 

With  the  apparatus  as  outlined  on  hand,  the 
serum  collected  as  described,  the  corpuscle  suspension 
freshly  prepared,  and  with  the  other  reagents  secured 
from  a  reliable  source,  the  test  can  be  carried  out  as 
will  be  described  in  the  following  pages. 

TECHNIC    OF    THE    TEST. 

To  facilitate  the  carrying  out  of  the  test  a  rack 
containing  two  rows  of  holes  for  the  tubes  as  shown 
in  the  illustration  on  page  64  should  be  used.  For 
each  test  two  tubes  are  required,  one  in  the  front  row 
and  its  control  in  the  rear  row.  There  will  also  be  two 
pairs  of  tubes  to  sers'^e  as  positive  and  negative 
controls. 

Put  into  each  of  two  small  test-tubes  front  and 
rear  one  drop  (0.02  c.c.)  of  the  serum  to  be 
tested  from  a  capillary  pipette.^  Add  to  each  tube 
0.1  c.c  of  40  per  cent,  fresh  guinea-pig  serum  ^  made 
by  adding  1  part  of  complement  to  1^^  parts  of  0.9 
per  cent,  salt  solution.  In  an  emergency  or  where 
fresh  complement  cannot  be  obtained  dried  slips  of 
paper  each  containing  two  units  of  complement  may 
be  substituted.  To  the  front  tube  add  the  slip  bearing 
the  antigen  in  form  of  an  emulsion  (recommended) 
or  of  an  impregnated  paper  slip.  Then  to  both  tubes 
add  1  c.c.  of  the  one  per  cent,  suspension  of  washed 

^When    using    inactivated    serum    put    4    drops     (0.08   c.c.)     into 
each  tube.     Use  0.2  c.c.  of  cerebrospinal  fluid  not  "  inactivated." 
^  See  under  Section  B,  p.  74. 


60  SERUM  DIAGNOSIS  OF  SYPHILIS. 

human  corpuscles  (the  first  procedure).  In  case  of 
using  the  ten  per  cent,  corpuscle  suspension  add  0.9 
c.c.  of  salt  solution  and  2  units  of  amboceptor  (the 
second  procedure) .  Shake  the  tubes  thoroughly  from 
time  to  time  to  distribute  the  reagents  evenly  through- 
out the  mixture. 

With  every  series  of  tests  it  is  necessary  to  carry 
out  two  sets  of  controls  as  already  referred  to  in 
beginning  the  description  of  procedure,  and  for  this 
purpose  four  additional  tubes  will  be  necessary.  To 
each  of  the  first  pair  of  these,  one  in  the  front  and 
one  in  the  rear  row,  one  capillary  drop  of  a  syphilitic 
serum  known  to  give  a  positive  reaction  is  added. 
This  will  serve  as  a  positive  control.  To  the  second 
pair  one  drop  of  normal  serum  known  to  give  a  nega- 
tive reaction  should  be  added,  or  the  tubes  may  be 
left  empty.  This  pair  of  tubes  will  serve  as  a  nega- 
tive control.  Now  put  into  each  tube  complement 
and  into  the  tubes  of  the  front  row  only  antigen, 
adding  finally  1  c.c.  of  the  one  per  cent,  corpuscle  sus- 
pension (first  procedure),  or  0.9  c.c.  of  salt  solution 
and  2  units  of  amboceptor  (second  procedure)  to 
each  tube. 

Place  the  rack  holding  these  pairs  of  tubes  in  a 
water-bath,  thermostat,  or  warm  place  not  over  37°  C. 
Allow  an  hour  from  the  time  the  mixture  is  made  for 
the  antibody  to  combine  with  the  antigen  and  for 


SERUM  DIAGNOSIS  OF  SYPHILIS.  61 

complement  to  be  fixed.  If  a  water-bath  is  used,  30 
minutes  is  a  sufficient  length  of  time.  If  dried  paper 
complement  is  used  this  period  of  incubation  should 
be  extended  to  twice  as  long  as  is  the  case  when 
Hquid  complement  is  used.  The  contents  of  the  tube 
in  the  first  procedure  are  as  follows: 

Rear:  Tvfist  ser.+ complm.  (2  units)+  O  +  1%  corp.  susp.  (1  c.c.) 
Front:  Test  ser.+  complm.    (2  units)  +  antigen  +  1%  corp.  susp.  (1  c.c.) 

The  contents  of  tubes  in  the  second  procedure  are 
as  follows: 

Rear:    Test  serum +complm.  (2  units)  +      O      +  amboceptor  (2  units). 
Front:  Test  serum +complm.  (2  units)  +  antigen  +  amboceptor  (2  units). 

First  incubation  at  37°  C.  for  1  hour,  then  add 
to  each  tube  of  the  first  procedure  a  slip  bearing  two 
units  of  amboceptor  as  shown  in  the  illustration,  as 
follows : 

Rear:     Above  +  amboceptor  (2  units). 
Front:   Above  +  amboceptor  (2  units). 

To  each  tube  of  the  second  procedure  0.1  c.c.  of 
the  ten  per  cent,  corpuscle  suspension,  as  follows : 

Rear:     Above  +  10%  corpuscle  suspension    (0.1   c.c). 
Front:  Above  +  10%   corpuscle  suspension    (0.1   c.c). 

AUow  another  two  hours  in  the  thermostat  or  one 
hour  in  water-bath.  After  final  incubation  the  tubes 
should  be  kept  at  room  temperature  for  a  few  hours 
before  the  results  are  recorded. 

In  accompanying  charts  various  steps  of  making 
the  test  by  the  first  and  second  procedures  are  given. 
After  the  above  stages  have  been  carried  out,  the 
result  of  the  reaction  can  be  read. 


SERUM  DIAGNOSIS  OF  SYPHILIS. 


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64i 


SERUM  DIAGNOSIS  OF  SYPHILIS. 


First,  it  is  necessary  to  make  certain  that  the 
tests  in  the  control  sets  have  been  properly  carried 
out.  The  pair  of  tubes  containing  normal  serum  (or 
without  any  serum)  must  be  completely  hsemolysed. 
These  constitute  the  negative  controls  and  show  that 
the  hemolytic  system  used  is  effective  (see  rear  tube) 


■Amboceptor 


Set  for  d/egnosii 


Antigen^  -Q 


^Antigen-  -O 


Fig.  10.— The  picture  shows  the  appearance  of  all  tubes  after  the  first  incubation  and 
just  at  the  moment  when  the  amboceptor  slip  is  about  to  be  added.  Thus  far  there  is 
no  visible  difierence  in  these  tubes  and  the  corpuscles  are  still  intact.  In  all  front  tubes 
there  are  small  square  pieces  of  paper  representing  the  antigen,  while  none  in  rear  tubes. 
These  square  slips  will  not  be  there  when  working  with  liquid  antigen. 

and  that  the  amount  of  antigen  used  is  not  by  itself 
inhibitory  of  haemolysis  (see  front  tube). 

Next,  the  front  tube  of  the  positive  control  set, 
containing  a  known  syphilitic  serum,  must  show  total 
inhibition  of  haemolysis,  while  the  rear  tube  must 
show  complete  haemolysis.     Thus  we  are  certain  by 


SERUM  DIAGNOSIS  OF  SYPHILIS. 


65 


the  rear  tube  that  the  syphihtic  serum  itself  does  not 
inhibit  hsemolysis,  while  the  front  tube,  in  which 
haemolysis  is  inhibited,  shows  the  abihty  of  syphilitic 
antibody  to  fix  complement  in  the  presence  of  the 
antigen  employed. 

These  essentials  having  been  fulfilled,  the  tubes 


•Jet  for  diagnosis. 


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fli Stp   <iii!;j_3iii>     «ii;i;_j' 


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V 


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Fig.  11. — This  picture  shows  the  appearance  of  the  tubes  after  completion  of 
haemolysis,  namely,  after  the  second  incubation.  In  all  front  tubes  there  are  two  square 
pieces  of  paper,  one  representing  antigen  and  the  other  amboceptor.  In  each  rear  tube 
there  is  but  one  piece,  and  it  represents  amboceptor.  In  negative  control  set  hsemolysis 
occurred  in  both  tubes.  In  positive  control  set  hsemolysis  took  place  in  the  rear  tube 
only  and  not  in  the  front.  In  the  set  for  diagnosis  the  conditions  are  seen  to  be  identi- 
cal with  the  positive  control  set,  hence  this  serum  is  found  to  be  syphilitic. 

containing  serum  for  diagnosis  can  be  scrutinized. 
In  these  tests  hsemolysis  must  be  complete  in  the  rear 
row,  since  antigen  is  not  present  and  the  amount  of 
serum  used  should  not  be  inhibitory.  Should  hsemol- 
ysis be  inhibited  markedly,  showing  usually  an  anti- 
complementary action  on  the  part  of  the  patient's 
serum,  this  may  be  overcome  by  "  inactivation  "  of 


66 


SERUM  DIAGNOSIS  OF  SYPHILIS. 


the  serum  for  20  minutes  at  56°  C  (before  com- 
mencing), after  which  it  will  be  necessary  to  use  4 
drops  of  the  serum  in  the  test.  The  above  irregu- 
larity is  occasionally  encountered  in  the  test,  espe- 
cially during  the  summer  months. 

Now,  the  tubes  containing  the  serum  for  diagnosis 


Fig.  12.— This  picture  shows  the  appearance  of  the  tubes  shown  in  Fig.  11  after 
standing  for  several  hours.  The  absence  of  haemolysis  in  the  front  tubes  of  positive 
control  and  diagnostic  sets  is  shown  by  clear  supernatant  salt  solution  over  the  deposited 
ntact  corpuscles.    The  absence  of  haemolysis  means  positive  reaction  in  these  instances. 

and  antigen,  the  front  row  of  tubes,  ma}''  be  examined 
for  final  results. 

Here  any  degree  of  hemolysis  ma}^^  be  encoun- 
tered, from  total  inhibition  to  complete  dissolution  of 
corpuscles,  depending  on  the  presence  or  absence  of 
syphilitic  antibodies  and  the  number  of  antibody 
units.  With  complete  inhibition  of  haemolysis,  the 
end  reaction  is  easily  interpreted,  the  corpuscles  set- 


SERUM  DIAGNOSIS  OF  SYPHILIS.  67 

tling  to  the  bottom  of  the  tube  with  the  clear  salt 
solution  above  (see  the  front  tube  of  the  pair  at  ex- 
treme left).  Complete  hasmolysis  likewise  gives  a 
result  easy  of  interpretation,  for  the  corpuscle  mass  is 
entirely  dissolved,  the  haemoglobin  going  into  solution 
and  coloring  the  salt  solution  a  deep  reddish  color 
(see  the  front  tube  of  the  pair  at  extreme  right). 

By  taking  into  consideration  the  bulk  of  cor- 
puscles settling  to  the  bottom  of  the  tube  and  the 
amount  of  tinting  of  the  supernatant  salt  solution, 
and  by  comparison  with  the  positive  and  negative  con- 
trols, the  varying  degrees  of  inhibition  of  haemolysis 
may  be  ascertained  (see  Figs.  11,  12  and  13).  In 
interpreting  the  result  complete  inhibition  of  haemol- 
ysis comparable  with  the  positive  control  is  called 
positive;  complete  haemolysis,  comparable  with  the 
negative  control,  is  designated  as  a  negative  reaction. 
If  60  to  70  per  cent,  of  the  bulk  of  corpuscles  is  dis- 
solved the  reaction  is  doubtful  and  should  not  be 
taken  into  consideration  for  diagnosis.  In  a  known 
specific  case  such  slight  inhibition  should  be  an  indi- 
cation for  further  treatment  as  evidence  of  the  con- 
tinued presence  of  syphilitic  antibodies  in  the  patient's 
blood.  If  there  is  a  faint  trace  of  heemolysis,  the  main 
bulk  of  corpuscles  being  intact,  the  reaction  should 
be  called  weakly  positive.  A  more  intense  haemolysis, 
with  about  10  to  20  per  cent,  dissolution  of  the  cor- 
puscle   mass,    should    be    called    very  weakly  post- 


68  SERUM  DIAGNOSIS  OF  SYPHILIS. 

tive^  while  30  to  40  per  cent,  hsemolysis  is  designated 
as  faintly  positive.  Neither  the  weakly  positive  nor 
the  faintly  positive  reaction  should  he  accepted  as  a 
definite  diagnosis  of  syphilis  without  the  presence  of 
strong  clinical  evidence  in  favor  of  such  a  diagnosis. 

In  case  the  reaction  should  be  doubtful,  the  serum 
should  be  re-examined  after  a  period  of  a  week  has 
elapsed,  and  if  necessary  several  examinations  in 
succession  should  be  made.  The  reaction  may  some- 
times be  very  weak  in  a  case  of  undisputed  syphilis. 
In  such  instance  the  test  should  be  made  not  only 
with  one  but  also  two  drops  at  the  same  time.  Very 
often  a  good  positive  reaction  can  be  obtained  with 
two  drops.  The  advantage  of  making  a  test  in  this 
class  of  cases  is  that  the  positive  finding  of  the  serum 
reaction  furnishes  the  clinician  with  one  more  sign 
of  the  existence  of  syphilitic  infection  besides  the 
other  visible  clinical  manifestations,  and  enables  him 
to  detect  the  persistence  or  cessation  of  the  active 
syphilitic  process  when  the  other  symptoms  have  dis- 
appeared. 

For  examining  the  sera  from  known  cases  of 
syphilis  for  prognostic  purposes  the  test  must  be 
made  with  two  drops  in  case  one  drop  gives  no 
longer  a  positive  reaction.  In  all  cases  it  is  a  wise 
precaution  to  take  the  blood  for  examination  shortly 
before  meal-time. 


HQ 


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SERUM  DIAGNOSIS  OF  SYPHILIS.  69 

It  may  be  stated  here  that  certain  specimens  of 
human  sera  gradually  become  anticomplementary 
after  several  days  on  standing,  some  more  pro- 
nouncedly so  than  others.  This  change  sets  in  much 
more  rapidly  at  a  higher  temperature,  say  that  of  a 
room,  than  at  a  lower  temperature,  say  of  a  refrigera- 
tor. 

In  order  to  have  a  positive  control  test  whenever 
examining  an  unknown  serum  one  must  always  have 
at  hand  a  syphilitic  serum  known  to  give  a  positive 
reaction.  For  this  purpose  one  has  to  obtain  a  good 
specimen,  which  can  be  preserved  on  ice  for  months. 
Should  such  a  specimen  become  too  anticomplemen- 
tary on  standing,  one  can  remove  this  property  by 
heating  the  serum  at  55^-56°  C.  for  about  fifteen 
minutes. 

TITRATION    OF    THE    ANTIBODY    CONTENT    OF 
SYPHILITIC    SERUM. 

In  the  routine  examination  of  patient's  serum  for 
the  presence  of  syphilitic  antibodies,  as  above  de- 
scribed, one  drop  (0.02  c.c.)  from  a  capillary  pipette 
is  used.  This  amount  of  serum  is  used  to  determine 
whether  the  serum  is  that  of  a  luetic  case  or  not.  When 
a  strong  positive  reaction  is  obtained  in  several  speci- 
mens we  are  unable  to  distinguish  the  intensity  of  the 
reaction  without  further  analysis.  In  fact,  a  positive 
reaction  may  be  gotten  with  any  syphilitic  serum  con- 


70  SERUM  DIAGNOSIS  OF  SYPHILIS. 

taining  many  units  of  syphilitic  antibody  or  that  quan- 
tity which  is  just  sufficient  to  deviate  complement.  A 
serum  may  thus  contain  more  than  one  unit  of  anti- 
body, and  in  order  to  determine  the  exact  strength 
of  each  specimen,  it  should  be  titrated  in  the  following 
way. 

Prepare  a  stock  dilution  of  the  serum  by  mixing 
0.1  c.c.  of  the  serum  with  4.9  c.c.  of  salt  solution. 
This  dilution  is  of  such  strength  that  1  c.c.  contains 
0.02  c.c.  of  the  original  serum  (corresponding  with 
one  drop  recommended  for  the  routine  test).  Thus 
1  c.c.  of  the  dilution  is  the  maximum  volume  of  fluid 
for  one  tube  and  contains  the  maximum  amount 
of  the  serum  chosen  for  the  test.  As  we  are 
going  to  titrate  the  strength  of  a  serum  giving 
a  positive  reaction  in  the  amount  of  0.02  c.c.  we  have 
to  test  different  amounts  of  the  same  serum  below 
0.02  c.c.  For  accomplishing  this  end  graded  quan- 
tities of  the  diluted  serum  are  measured  into  a  number 
of  tubes,  as  shown  in  the  following  protocol : 


Dilution 

OF 

Serum. 

Tube  1.... 

....1 

c.c. 

(equal 

to  0.02     c.c. 

original  serum) 

2. . . . 

....0.5 

"  0.01 

a                 a 

3.... 

....0.4 

"  0.008 

"                  " 

4.... 

....0.3 

"  0.006 

CC                          C( 

5.... 

....0.25 

"  0.005 

<C                          (( 

6.... 

....0.2 

"  0.004 

cc                    « 

7.... 

0.165 

( 

"  0.0033 

C>                          (f 

8.... 

....0.125 

"  0.0025 

It                « 

9.... 

....0.1 

"  0.002 

cc                      It 

SERUM  DIAGNOSIS  OF  SYPHILIS.  71 

Having  measured  the  amounts  of  the  serum  into 
the  tubes  and  bringing  the  total  volume  of  fluid 
of  each  tube  up  to  1  c.c.  uniformly  by  adding  salt 
solution,  one  now  proceeds  to  make  the  fixation  test 
in  the  usual  way.  Add  to  every  tube  0.1  c.c.  of  40 
per  cent,  dilution  of  guinea-pig's  complement,  0.1 
c.c.  of  the  standard  antigen  emulsion  and  2  units  of 
amboceptor.  Mix  the  content  well  by  shaking  and 
incubate  the  tubes  at  37° C.  for  one  hour.  At  the 
end  of  the  incubation  add  to  every  tube  0.1  c.c.  of 
10  per  cent,  suspension  of  washed  human  corpuscles, 
and  incubate  at  37° C.  for  two  hours.  The  results 
are  then  read.  According  to  the  strength  of  serum 
in  question  the  complete  inhibition  of  haemolysis  may 
occur  in  any  of  the  tubes  in  the  series.  If  a  given 
specimen  should  give  complete  fixation  in  the  first 
tube  (1  c.c),  it  would  be  said  to  contain  only  one 
unit  of  antibody.  If  this  occurs  in  the  second  tube 
(0.5  c.c.)  it  contains  two  units;  in  the  third  (0.4  c.c), 
2.5  units;  in  the  fourth  (0.3  c.c),  3.3  units,  etc.  A 
specimen  showing  complete  fixation  in  the  last  tube 
(0.1  c.c.)  must  contain,  at  least,  ten  antibody  units. 
If  the  serum  gives  a  strong  fixation  in  the  last  tube 
(0.1  c.c.)  it  is  best  to  titrate  it  with  a  second  dilution 
in  order  to  find  out  its  real  titre. 

By  this  means  the  titre  of  any  specimen  can  easily 
be  determined.  The  writer  has  examined  specimens 
which  contained  as  many  as  20  units. 


72  SERUM  DIAGNOSIS  OF  SYPHILIS. 

In  case  of  titrating  cerebrospinal  fluid  one  must 
prepare  the  stock  dilution  by  mixing  1  c.c.  of  it  with 
4  c.c.  of  salt  solution.  The  process  of  titration  is 
exactly  the  same  as  that  described  for  the  serum. 

In  titration  work  graduated  pipettes  are  to  be 
employed. 

Section  B. 

methods  of  preparing  the  reagents  and  of 

making  a  complete  fixation  test  with 

preparations  of  unknown  value. 

COMPLEMENT. 

The  views  of  the  author  regarding  the  use  of 
complement  dried  in  paper  have  been  modified 
soon  after  its  introduction,  and  now  he  considers 
it  advisable  to  use  the  liquid  complement  whenever 
possible.  While  it  is  quite  possible  to  prepare  the 
complement  on  paper  its  use  should  be  reserved  for 
emergencies — when  the  fresh  complement  cannot  be 
obtained. 

Guinea-pig's  serum  is  to  be  used.  Large  animals 
are  selected  and  bled  by  cutting  the  carotid  artery, 
allowing  the  blood  to  flow  into  a  large  Petri  dish.  The 
dish  is  then  covered  and  left  at  room  temperature  for 
a  few  hours  for  the  clot  to  form  and  the  serum  to 
separate.  Then  the  separation  of  the  serum  may  be 
completed  in  the  refrigerator.  Within  five  to  ten 
hours  all  the  serum  has  separated  from  the  clot  and 


SERUM  DIAGNOSIS  OF  SYPHILIS.  73 

should  then  be  poured  into  a  sterile  test-tube  and 
thereafter  when  not  in  use  kept  in  the  refrigerator. 
No  preservative  may  be  added.  After  the  serum  is 
48  to  72  hours  old  the  activity  of  complement  is 
rapidly  lost,  even  at  refrigerator  temperature,  if  the 
serum  is  kept  in  a  fluid  form. 

The  author  does  not  advise  the  use  of  complement 
dried  on  paper  if  it  is  at  all  possible  to  obtain  fresh 
guinea-pig's  serum.  However,  mindful  of  the  fact 
that  this  is  difficult  if  not  impossible  of  accomphsh- 
ment  under  certain  circumstances,  as,  for  example, 
in  out-of-the-way  places,  army  camps,  etc.,  the  method 
of  preparing  complement  paper  will  be  described. 

Preparation  of  Complement  Slips. — Squares  of 
thick  blotting-paper  are  put  in  a  sterile  flat  dish  and 
serum  is  poured  over  it  until  the  paper  is  thoroughly 
soaked  and  an  excess  remains.  The  saturated  paper 
is  then  removed  to  another  dish  or  flat  tray  and  quickly 
dried  in  a  current  of  air  at  a  temperature  not  above 
10°  C,  the  lowest  possible  temperature  being  the 
most  useful.  The  drying  should  be  accomplished  with- 
in an  hour.  According  to  the  thickness  of  the  paper  a 
second  impregnation  is  recommended.  After  com- 
plete desiccation  the  paper  is  standardized  in  the  fol- 
lowing way:  Use  a  hsemolytic  system  composed  of 
human  erythrocytes  and  an  antihuman  amboceptor. 
The  erythrocyte  suspension  is  to  be  that  used  in 


74  SERUM  DIAGNOSIS  OF  SYPHILIS. 

our  test,  that  is,  1  c.c.  of  one  per  cent,  suspension 
of  human  corpuscles.  The  amboceptor  must  have 
been  first  standardized  with  fresh  fluid  guinea-pig 
complement.  Arrange  a  series  of  tubes  each  con- 
taining 1  c.c.  of  one  per  cent,  erythrocyte  suspension 
and  one  unit  of  amboceptor.  Now,  selecting  a  sample 
of  the  paper  impregnated  with  complement,  cut  it 
into  strips  of  a  given  dimension  in  millimetres, 
preferably  5  mm.  wide.  Add  to  the  series  of  tubes 
bits  of  paper  of  increasing  length,  say  2,  3,  5,  7,  10, 
15  mm.,  etc.  Incubate  the  mixture  at  37°  C.  for  two 
hours.  That  size  of  slip  in  which  haemolysis  is  just 
complete  will  contain  one  unit  of  complement.  As 
we  desire  to  use  two  units  of  complement  in  the  fixa- 
tion test,  the  remaining  paper  will  be  measured  off 
into  squares  having  twice  the  dimension  of  that 
found  for  one  unit.  These  may  be  marked  with 
pencil  so  that  they  may  be  snipped  off  as  they  are 
needed.  These  strips  must  be  kept  perfectly  dry  and 
sealed  hermetically  in  vacuum. 

PREPARATION  OF  AMBOCEPTOR. 

Antihuman  hsemolytic  amboceptor  is  to  be  used. 
This  is  made  by  immunizing  rabbits  against  human 
blood-corpuscles.  Select  large  rabbits  and  inject 
increasing     amounts     of    washed  ^     human    blood- 

^  The  corpuscles  must  be  washed  at  least  three  times  with  a  large 
amount  of  saline  solution.  If  this  is  not  done  the  immune  serum  may 
contain  precipitin  for  human  serum,  which  will  interfere  with  the  fixa- 
tion reaction. 


SERUM  DIAGNOSIS  OF  SYPHILIS.  76 

corpuscles  five  times  in  succession  intraperitoneally, 
allowing  a  four-  or  five-day  interval  between  injec- 
tions.^ Nine  or  ten  days  after  the  last  injection,  bleed 
the  rabbit  from  the  carotid  artery  with  a  blood-tube 
shown  in  the  drawing  on  page  76. 

Schedule   for   Immunization. 

Injections  at  four-  or  five-day  intervals.     Bleeding  nine  or  ten  days 
after  the  last  injection. 

1st  injection,     5  c.c.  of  the  washed  human  corpuscles. 

2d    injection,     8  c.c.  of     "           "            "  " 

3d    injection,  12  c.c.  of     "           "            "  " 

4th  injection,  15  c.c.  of     "           "            "  " 

5th  injection,  20  c,c.  of     "           "            "  " 

After  the  blood  is  collected  the  blood-tube  is 
placed  at  room  temperature  for  several  hours  before 
being  transferred  to  the  refrigerator.  During  this 
period  the  clot  gradually  contracts  and  separates  from 
the  wall  of  the  tube,  allowing  a  clear  serum  to  exude 
in  the  space  between  the  clot  and  the  wall  of  the  tube. 
If  the  clot  remains  uncontracted  within  four  or  five 
hours,  separate  it  carefully  from  the  wall  by  insert- 
ing a  sterile  stiff  platinum  needle  or  glass  rod,  and 
allow  it  to  stand  at  room  temperature  for  several 
hours  again  to  promote  the  contraction  of  the  clot. 
Then  place  the  blood  in  a  refrigerator  for  twenty- 
four  hours.     Collect  the  clear  serum  at  the  end  of 

1  Four  intravenous  injections,  4  c.c,  3  cc,  4  c.c,  3  c.c  and  pos- 
sibly another  4  c.c.  with  four-  or  five-day  intervals,  give  also  good  results. 
This  mode  of  immunization  is,  however,  less  safe  for  the  rabbits. 


76 


SERUM  DIAGNOSIS  OF  SYPHILIS. 


twenty-four  hours  by  decantation,  and  leave  the  tube 
for  another  day  to  collect  serum  again.    Repeat  this 


S/ssi  Csnuh 

Jncaie  afta/ringlhg 
blood  from  a  ire  in  of 
man  fhispsrt  must 
be  substituted  mth 
a  regular  syringe 
neectk.) 


-  —Jim//ie.}{  tube 


Fig.  14. — Sterilize  in  steam,  but  not  in  hot  air.  For  the  purpose  of  taking  the 
blood  from  man  the  size  of  the  tube  should  be  that  of  ordinary  test-tube  and  should  be 
provided  with  a  good-sized  syringe  needle  instead  of  a  glass  canula. 

*This  can  be  smaller,  although  less  convenient  than  a  larger  one. 

for  three  or  four  successive  days,  until  no  more  serum 
is  given  out  by  the  clot  on  further  standing.  The 
portions  of  the  serum  collected  in  this  manner  over 


SERUM  DIAGNOSIS  OF  SYPHILIS.  77 

several  days  may  be  mixed  together.  When  the  serum 
contains  a  certain  amount  of  corpuscles  let  it  stand 
for  a  day  or  longer  in  an  ice-box  to  allow  the  latter 
to  sink  to  the  bottom,  and  collect  the  clear  serum  by 
gentle  decantation,  or  centrifugalization  of  the  bloody 
serum  may  be  resorted  to. 

The  amboceptor  had  best  be  titrated  in  fluid  form 
when  collected  to  be  sure  it  is  strong  enough,  before 
going  to  the  trouble  of  impregnating  paper  with  it.^ 
The  principles  of  the  titration  were  fully  discussed 
in  Chapter  II.  A  good  preparation  will  have  a 
value  of  1  unit  in  something  less  than  0.001  c.c.  of 
serum,  that  is,  0.001  c.c.  of  serum  or  less  will  cause 
complete  hgemolysis  of  1  c.c.  of  a  one  per  cent,  sus- 
pension of  human  erythrocytes  when  combined  with 
0.02  c.c.  of  guinea-pig's  fresh  serum  (0.1  c.c.  of  20 
per  cent,  dilution). 

Method  of  Preparing  Amboceptor  Slips. — When 
it  has  been  determined  that  our  preparation  is 
of  the  required  strength,  as  will  usually  be  the  case 
if  the  method  of  immunization  outUned  is  followed, 
we  proceed  to  the  preparation  of  the  paper.  This  is 
a  more  simple  matter  in  the  case  of  the  amboceptor 
than  in  the  case  of  complement,  for  the  former  is 
stable.    As  our  slip  will  only  contain  about  0.001  c.c. 

^  Diiferent  rabbits  react  differently  to  amboceptor  production  and  it 
is  not  seldom  to  get  a  weak  serum  after  a  long  immunization,  while  a 
powerful  serum  may  be  gotten  after  four  injections. 


78  SERUM  DIAGNOSIS  OF  SYPHILIS. 

of  serum  in  place  of  0.04  c.c,  a  thinner  paper  is  used. 
I  have  found  Schleich  &  Schull's  paper  ISTo.  597  satis- 
factory.    The  paper  is  cut  into   squares  of  about 
10  X  10  cm.  and  soaked  with  the  serum  in  the  same 
general  way  as  is  the  complement,  but  here  I  avoid  too 
great  an  excess,  seeking  only  to  get  all  the  sheets 
evenly  wet,  then  absorbing  the  excess  with  another 
sheet  of  paper.    The  paper  can  then  be  dried  at  room 
temperature  by  placing  each  square  separately  upon  a 
clean  sheet  of  non-absorbent   (unbleached)    muslin. 
Several  hours'  drying  usually  suffices.     The  sheets 
when  thoroughly  dry  are  cut  into  convenient  width, 
say  5  mm.,  and  then  standardized.    Take  a  series  of 
tubes  containing  one  cubic  centimetre  of  the  one  per 
cent,  erythrocyte  suspension  as  described  for  the  titra- 
tion of  complement.    Add  to  each  definite  amount  of 
complement  (0.02  c.c.)  as  one  unit.    Then  add  to  the 
series  measured  increasing  lengths  of  the  amboceptor 
strip,  e.g. J 1  mm.,  2  mm.,  3  mm.,  etc.,  and  incubate  the 
series  for  two  hours.    The  shortest  strip  which  causes 
complete  haemolysis  in  this  time  contains  1  ambocep- 
tor unit.     The  strips  are  then  marked  into  sections 
of  ttJDice  this  length  and  cut  off  at  the  time  of  doing 
the  test.    Each  section  will  then  contain  the  two  units 
to  be  used  in  the  test.    These  papers  should  be  kept 
dry  and  sealed,  but  the  same  extreme  precautions  need 
not  be  taken  with  them  as  with  the  complement  paper. 


SERUM  DIAGNOSIS  OF  SYPHILIS.  79 

PREPARATION    OF    ANTIGEN. 

It  is  settled  to-day  that  alcoholic  extracts  of 
certain  tissues  contain  variable  quantities  of  "  anti- 
gen "  for  syphilis.  There  is  more  in  heart,  liver, 
or  kidney  than  in  nervous  tissues,  so  far  as  has 
been  determined.  The  liver  of  a  congenitally 
syphilitic  foetus  was  once  considered  as  one  of  the 
tissues  richest  in  these  antigenic  lipoids,  although 
this  claim  became  no  longer  tenable  through  later 
investigations.  I  have  found  that  not  only  selected 
samples  of  tissue  lecithin,  but  also  several  other  phos- 
phatids,  as  well  as  several  aceton-soluble  fractions 
of  tissue  lipoids,  can  act  as  antigen.  Whether  the 
extract  derives  from  syphilitic  organs  or  from  non- 
syphilitic  tissues  makes  but  little  difference.  Animal 
tissues  are  just  as  good  as  human  in  point  of  yielding 
a  serviceable  extract.  In  all  cases  one  has  to  deter- 
mine the  extract  before  using  it  as  antigen.  It  is 
possible  to  obtain  good  preparations  from  animal 
tissues  just  as  frequently  as  from  human  organs. 
The  method  of  preparing  the  antigenic  extract  will 
be  given  below. 

Extract  a  mashed  paste  of  liver,  heart,  or  kidney 
of  man,  ox,  guinea-pig,  rabbit,  or  dog  with  10  parts 
of  absolute  alcohol  at  37°  C.  for  several  days.  Filter 
through  paper  and  collect  the  filtrate.  The  latter 
is  then  brought  to  dryness  by  evaporation  with  the 


80  SERUM  DIAGNOSIS  OF  SYPHILIS. 

aid  of  an  electric  fan.  The  residue  is  then  taken  up 
with  a  sufficient  quantity  of  ether  and  the  turbid 
ethereal  solution  is  allowed  to  stand  for  over  night  in 
a  cool  place ;  the  receptacle  must  be  covered  in  order 
to  prevent  evaporation  of  ether  during  this  period. 
The  next  morning  one  will  find  that  the  turbidity  is 
entirely  cleared  up  by  gravitation  of  the  insoluble 
particles  to  the  bottom.  The  clear  ethereal  portion  is 
then  carefully  decanted  into  another  clean  beaker  and 
condensed  into  a  small  quantity  by  evaporating  the 
ether  off.  The  concentrated  ethereal  solution  is  now 
mixed  with  about  ten  volumes  of  pure  aceton. 

A  precipitate  forms,  which  is  allowed  to  settle  to 
the  bottom  of  the  vessel,  and  the  supernatant  fluid 
decanted  off.  We  thus  obtain  a  light  brownish  pre- 
cipitate which  gradually  becomes  sticky  on  exposure 
to  the  air.  This  aceton-insoluble  portion  of  the  tissue 
extract  contains  antigenic  lipoids  and  its  quality  and 
strength  must  be  determined. 

Titration  of  Antigen. — In  order  to  ascertain 
whether  a  given  extract  is  suitable  for  antigen  or  not 
an  emulsion  is  first  prepared  and  tested.  If  it  is 
found  suitable  the  extract  may  be  made  an  alcoholic 
stock  solution  and  an  emulsion  from  this  be  prepared 
at  the  time  of  making  the  test,  or  it  may  be  impreg- 
nated in  paper  and  used  in  the  form  of  dried  strips. 
I  will  describe  here  the  general  way  of  determining 
the  antigenic  value  of  a  given  extract. 


SERUM  DIAGNOSIS  OF  SYPHILIS.  81 

Before  entering  into  technical  details  of  titration 
of  the  antigenic  lipoids  certain  properties  of  this 
fraction  in  general  may  be  dealt  with  here.  Usually 
the  aceton-insoluble  fraction  of  tissue  lipoids  as  pre- 
pared by  the  method  just  described  has  no  hcemolytic 
action  upon  human  erythrocytes.  But  one  occasion- 
ally encounters  a  preparation  which  causes  haemolysis 
in  a  large  amount.  The  presence  of  such  an  injurious 
property  in  the  preparation  intended  for  antigen 
renders  it  unsuitable  and  it  should  be  discarded. 
For  this  reason  it  is  necessary  with  every  sample  of 
extract  to  test  it  for  heemolytic  property.  Another 
property  often  possessed  by  this  fraction  and  render- 
ing it  unsuitable  for  antigen  is  the  anticomplementary 
action^  namely,  the  property  to  diminish  or  destroy 
the  activity  of  complement.  In  a  very  large  amount 
this  anticomplementary  effect  may  be  manifest  in 
nearly  50  per  cent,  of  different  samples.  Therefore, 
we  must  examine  every  specimen  for  its  anticomple- 
mentary activity.  If  it  should  exceed  a  certain  limit 
the  specimen  is  not  to  be  employed  as  antigen. 

These  two  properties,  hsemolytic  and  anticomple- 
mentary, whether  present  simultaneously  or  singly, 
disqualify  a  given  sample  of  the  extract  as  a  reliable 
antigen  for  the  test.  After  eliminating  those  prepa- 
rations which  are  either  haemolytic  or  anticomple- 
mentary or  both  by  properly  arranged  series  of  deter- 


82  SERUM  DIAGNOSIS  OF  SYPHILIS. 

mination  one  at  last  takes  up  the  question  of  antigenic 
property.  By  antigenic  property  is  meant  that  which 
produces  fixation  of  complement  in  the  presence  of 
syphilitic  serum.  This  most  important  property  of 
the  extract  is,  however,  quite  variable  according  to 
different  samples.  Some  may  possess  exceedingly 
powerful  antigenic  action,  while  others  may  not  be 
antigenic  at  all.  Fortunately,  according  to  my 
recent  systematic  analysis  of  nearly  100  different 
specimens,  with  Mr.  Bronfenbrenner,  about  50  per 
cent,  were  found  to  be  serviceable  and  only  about  5 
per  cent,  were  devoid  of  this  property.  This  latter 
were  all  from  fatty  livers.  The  rest  of  the  specimens 
were  not  perfect  on  account  of  the  pronounced  anti- 
complementary combined  with  weaker  antigenic 
property. 

As  will  be  seen  from  the  above  general  presenta- 
tion of  the  subject  a  titration  of  antigen  really 
includes  the  determinations  of  (a)  hsemolytic,  (b) 
anticomplementary,  and  (c)  antigenic  activities. 
This  can  be  made  simultaneously  in  a  properly 
arranged  series  of  experiments.  The  following  is  the 
method  which  I  recommend  for  adoption  by  those 
who  employ  my  system: 

Stock  Solution  of  Antigen. — Take  0.3  gram  of 
the  aceton-insoluble  fraction  and  dissolve  in  about  1 
c.c.  of  ether  in  a  test-tube.     The  ethereal  solution  is 


SERUM  DIAGNOSIS  OF  SYPHILIS.  83 

then  mixed  with  9  c.c.  of  methyl  alcohol  in  which  the 
greater  part  of  substances  goes  into  solution.  The 
alcoholic  solution,  which  contains  3  per  cent,  of  the 
lipoids,  remains  unaltered  for  a  long  time  and  can 
be  kept  as  stock  from  which  the  emulsion  for  immedi- 
ate use  may  be  prepared  at  any  time. 

Emulsion  of  Antigen. — This  is  quickly  prepared 
by  mixing  up  1  c.c.  of  the  alcoholic  stock  solution  (see 
above)  with  9  c.c.  of  salt  solution.  The  emulsion 
thus  prepared  is  a  clear  opalescent  fluid.  It  is  this 
form  in  which  antigen  is  most  certain  in  action  and 
convenient  for  use.  The  concentration  of  the  emul- 
sion is  0,3  per  cent,  of  the  original  lipoidal  substances. 

METHOD  OF  SELECTING  A  SUITABLE  ANTIGEN. 

In  determining  whether  a  given  specimen  of 
extract  is  suitable  for  use  or  not  we  must  examine  it 
for  hsemolytic,  anticomplementary  and  antigenic 
jjroperties  as  already  discussed  in  the  beginning. 
When  a  specimen  does  not  show  either  hsemolytic  or 
anticomplementary  action  in  a  certain  quantity  it  is 
eligible  for  the  final  examination  for  antigenic 
strength.  It  is  found  through  our  long  experience 
and  experiments  that  the  quantity  of  emulsion  for 
testing  hsemolytic  and  anticomplementary  properties 
should  be  0.4  c.c,  and  that  for  testing  antigenic  power 
0.02  c.c.  In  measuring  such  a  small  quantity  as 
0.02  c.c.  of  emulsion  it  is  customary  first  to  prepare 
a  ten-fold  dilution  of  the  usual  emulsion  with  salt 


84  SERUM  DIAGNOSIS  OF  SYPHILIS. 

solution  and  measure  out  0.2  c.c.  of  the  dilution  (cor- 
responding with  0.02  c.c.  of  the  regular  emulsion). 
In  the  following  chart  I  present  the  processes  of 
determination. 

Chart  1. 


Tube  1  (Test  for  hsemolytic  property). 


Antigen   emulsion    0.4  c.c. 

Salt    solution     0.6  c.c. 

Corpuscle  suspension   (10%) 0.1  c.c. 


Incubation  at  37  °C.  for  2  hours. 


Tube  2  (Test  for  anticomplement  property) . 


Antigen   emulsion    0.4  c.c. 

Salt    solution    0.6  c.c. 

Complement    (40%)    0.1  c.c. 

Amboceptor   2   units 


Incubation  at  37  °C.   for  1  hour. 


Corpuscle  suspension  (10%)  0.1  c.c. 


Incubation  at  37  °C.  for  2  hours. 


Tube  3  (Test  for  antigenic  property) . 


Antigen  emulsion    (1 :10) 0.2     c.c. 

Salt  solution   0.8    c.c. 

Syphilitic  serum    0.02  c.c* 

Complement    (40%) 0.1     c.c. 

Amboceptor  2   units 


Incubation  at  37  °C.  for  1  hour. 


Corpuscle  suspension  (10%)  0.1  c.c. 


Incubation  at  37°C.  for  2  hours. 


One  drop  from  a  capillary  pipette. 


SERUM  DIAGNOSIS  OF  SYPHILIS.  85 

These  three  determinations  can  be  made  at  the 
same  time  in  a  series  of  experiments;  and  it  is  not 
necessary  to  wait  the  result  of  one  determination 
before  commencing  the  others. 

The  results  of  determinations  vary,  of  course, 
according  to  the  quality  of  the  specimen  under  con- 
sideration, and  one  may  obtain  any  one  of  the  possi- 
bilities shown  on  page  86. 

Now  scrutinizing  the  quantitative  relationship 
between  the  amount  of  emulsion  employed  for  test- 
ing the  hsemolytic  and  anticomplementary  properties 
and  that  for  antigenic  property  one  will  notice  that 
it  stands  20: 1.  This  renders  the  fixation  test  abso- 
lutely free  from  any  possible  interference  by  the  anti- 
complementary or  heemolytic  activity  of  the  antigenic 
preparation  alone.  It  may  not  be  amiss  to  remark 
at  this  place  about  the  requirements  laid  by  Wasser- 
mann  in  selecting  a  preparation  of  extract  for  the 
test.  He  made  it  the  rule  that  one  first  determines 
the  minimal  quantity  of  extract  which  inhibits 
haemolysis  and  then  test  if  the  half  of  this  anticom- 
plementary dose  gives  a  complete  fixation  with  a 
syphilitic  serum  or  not.  If  it  does,  the  preparation  is 
considered  suitable.  As  one  can  easily  understand 
this  quantity  of  extract  may  contain  just  one  antigen 
unit  or  several.  One  simply  goes  on  with  the  fixation 
test  without  knowing  how  many  antigen  units  he  is 
employing.    If  the  results  obtained  with  one  unit  of 


86 


SERUM  DIAGNOSIS  OF  SYPHILIS. 


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SERUM  DIAGNOSIS  OF  SYPHILIS.  87 

antigen  are  the  same  as  those  obtained  with  several 
there  would  be  no  chance  of  obtaining  variable  results, 
but,  in  reality,  the  reactions  vary  considerably  accord- 
ing to  whether  one  uses  one  or  several  antigen  units. 
This  is  especially  so  with  a  weaker  syphilitic  serum. 
In  my  opinion  it  is  absolutely  necessary  to  use  several 
antigen  doses  in  order  to  get  uniform  results  with  any 
specimen,  because  the  weakest  reaction  escapes  detec- 
tion, unless  one  employs,  at  least,  over  four  antigen 
units.  It  is  understood,  however,  that  no  oversensi- 
tive reaction  ought  to  be  allowed  to  occur  in  using 
many  antigen  units.  Fortunately,  according  to  our 
extensive  experience,  the  use  of  a  suitable  antigen — 
prepared  by  my  method — causes  no  such  an  undue 
sensitiveness. 

One  might  think  that  the  same  principle  can  be 
applied  also  to  the  Wassermann  system,  but  this  is 
impossible  because  of  the  constant  presence  of  anti- 
complementary substances  in  the  extract  used  in  that 
system. 

The  Quantity  of  Antigen  for  Fiocation  Test. — As 
stated  above  it  is  essential  for  obtaining  uniform 
results  to  employ  more  than  four  antigen  units.  To 
fulfil  this  requirement  I  recommend  the  use  of  0.1 
c.c.  of  0.3  per  cent,  suspension  of  the  aceton-insolu- 
ble  tissue  lipoids  whose  suitableness  as  antigen  has 
previously  been  determined  in  the  manner  already 
described.    In  this  quantity  of  the  emulsion  there  will 


88  SERUM  DIAGNOSIS  OF  SYPHILIS. 

be  at  least  five  antigen  doses  (0.1  c.c.  -h  0.02  c.c.  =  5) . 

Preservation  of  Antigen. — When  one  secures  a 
suitable  specimen  of  aceton-insoluble  tissue  lipoids  it 
may  be  preserved  in  the  following  manner. 

(a)  In  Solid  Form. — Put  up  the  solid  substance 
in  a  tube  and  seal  it  hermetically.  It  is  best  to  put  a 
small  quantity  of  aceton  to  the  tube  before  sealing. 

(h)  In  Alcoholic  Solution. — Dissolve  0.3  gram 
in  small  amount  of  ether  and  then  add  10  c.c.  of 
methyl  alcohol.  The  solution  is  then  divided  into 
small  portions  (say  each  1  c.c.)  and  sealed  in  ampoules. 
In  time  of  use  take  1  c.c.  of  this  stock  solution 
and  mix  with  9  c.c.  of  salt  solution,  thus  making  the 
0.3  per  cent,  emulsion  ready  for  use.  When  emulsi- 
fied the  antigen  should  be  kept  on  ice;  it  is  not  very 
stable  in  this  form.  Therefore  it  is  best  to  prepare 
just  so  much  emulsion  each  time  as  may  be  used  up 
in  a  week  or  so,  and  then  a  fresh  lot  of  emulsion 
is  again  prepared.  The  alcoholic  stock  solution  may 
be  made  at  any  time  from  the  solid  lipoidal  substances 
preserved  in  the  manner  described  under  (a).  The 
writer  found  that  the  mode  of  preservation  above 
stated  was  the  best  and  most  reliable. 

(c)  The  Antigen  Paper  Slips. — The  antigenic 
lipoids  may  be  impregnated  in  filter  paper  and  used 
in  form  of  dried  slips.  The  impregnated  paper  retains 
its  antigenic  property  for,  at  least,  three  months. 
Within  six  months  it  is  apt  to  deteriorate,  being  far 


SERUM  DIAGNOSIS  OF  SYPHILIS.  89 

less  durable  than  the  alcoholic  solution,  but  more 
stable  than  in  form  of  an  emulsion. 

The  process  of  impregnating  filter  paper  with  an- 
tigen is  similar  to  that  used  for  amboceptor.  Here 
we  use  ethereal  solution  of  the  antigenic  lipoids  for 
impregnation. 

Method  of  Preparing  Antigen  Slips. — Weigh 
out  about  1.2  grams  of  the  sticky  mass  of  the  ex- 
tract and  dissolve  in  about  20  c.c.  ether.  Have  ten 
sheets  of  filter  paper  of  the  dimensions  of  10  x  10 
cm.  ready,  laid  one  upon  the  other  in  a  clean  glass 
dish.  Pour  over  these  the  lipoid  solution  and  satu- 
rate the  paper  evenly.  Separate  each  sheet  as  quickly 
as  possible  and  lay  flat  on  a  clean  sheet  of  unbleached 
muslin,  as  in  the  case  of  the  amboceptor  paper. 
Evaporation  of  the  solvent  usually  takes  place  very 
quickly  and  within  ten  minutes  the  impregnated  paper 
is  ready  for  use.  Before  assigning  the  dimension  for 
each  tube  in  the  fixation  test  the  antigen  paper  should 
be  titrated.  This  is  done  in  the  following  manner. 
Cut  the  paper  into  equal  width,  say  5  mm.,  and  use 
increasing  lengths  of  this  strip  for  standardization, 
starting  with  1  mm.,  2  mm.,  3  mm.,  etc.  The  principle 
of  standardization  of  antigen  slips  is  the  same  as  de- 
scribed for  the  liquid  preparation,  differing  only  in 
using  paper  instead  of  liquid.  The  strips  may  be 
marked  in  sections,  each  representing  the  required 
dimensions,  and  put  into  sealed  tubes  for  preserva- 
tion. 


VIII. 

ADJUSTABILITY  OF  THE  WRITER'S  SYSTEM. 

Under  ordinary  circumstances  the  relative  quan- 
tities of  the  diiFerent  factors  prescribed  in  my  system 
should  give  uniform  and  reliable  results;  but,  as  the 
properties  of  the  complement  and  the  resistance  of  the 
red  corpuscles  sometimes  vary  according  to  their 
source  and  age,  certain  irregularities  now  to  be  con- 
sidered sometimes  arise.  In  view  of  these  variables 
it  is  desirable  that  the  worker  understand  how  to  ad- 
just relatively  the  quantities  of  these  factors.  Indeed, 
there  are  no  difficulties  that  can  arise  which  cannot 
be  removed  by  the  proper  use  of  the  several  reagents. 

1.  One  sometimes  meets  with  instances  in  which 
the  hfemolysis  is  complete  within  10  to  20  minutes, 
and  in  which  the  positive  control  tubes  with  antigen 
undergo,  sooner  or  later,  gradual  hgemolysis.  Such 
rapid  progress  of  haemolysis  at  first  mentioned  is  a 
sign  of  imperfect  reaction.  If  the  test  is  properly 
made,  haemolysis  proceeds  gradually,  and  is  complete 
in  the  water-bath  within  half  an  hour  or  thereabout. 
The  causes  of  this  accelerated  hgemolytic  process  are 
either  an  abnormally  weak  resistance  of  the  blood-cor- 
puscles, or  an  exceptionally  high  activity  or  insensi- 
tiveness  to   fixation   of  the   complement   employed; 

90 


SERUM  DIAGNOSIS  OF  SYPHILIS.  91 

or  it  may  be  the  result  of  all  these  acting  to- 
gether/ The  corjDuscles  should  never  be  older  than 
72  hours,  and  should  be  kept  constantly  on  ice, 
except  when  being  used  for  the  test.  They  decrease 
rapidly  in  strength  after  the  72-hour  period,  and 
more  quickly  if  kept  at  room  temperature.  Hav- 
ing even  chosen  suitable  corpuscles,  the  haemolysis 
may  still  proceed  too  rapidly,  in  which  case  the  com- 
plement is  likely  to  be  at  fault.  It  happens  occa- 
sionally that  the  serum  of  certain  guinea-pigs  contains 
an  abnormally  active  complement.  In  order  to  estab- 
lish this  point,  and  thus  to  remove  this  source  of  error, 
one  has  only  to  make  the  test  with  a  smaller  quantity 
of  the  complement,  or,  speaking  more  correctly,  a 
quantity  that  corresponds  exactly  to  two  complement 
units. 

2.  There  are  sometimes  encountered  instances  in 
which  hsemolysis  remains  incomplete  even  in  the 
control  tubes  in  which  there  is  no  antigen.  Here  the 
causes  of  the  imperfect  reaction  are  found  either  in 
the  weakness  of  the  complement,  or  the  amboceptor 
used,  or  both.  Usually  the  cause  is  the  weakness  of 
the  complement,  which,  owing  to  its  great  lability,  is 
likely  to  deteriorate.  Thus  it  is  a  good  practice  to 
employ  complement  that  is  not  older  than  48  hours, 
and  that  has  been  kept  constantly  at  refrigerator 

^  Guinea-pig's  complement  may  sometimes  remain  unfixed  and  mask 
the  positive  reaction.  It  is  always  best  to  use  a  mixture  of  the  sera 
from  two  or  more  guinea-pigs. 


92  SERUM  DIAGNOSIS  OF  SYPHILIS. 

temperature.  No  attempt  should  be  made  to  utilize 
a  deteriorated  complement  in  larger  quantities,  be- 
cause such  a  specimen  does  not  give  a  reliable  reaction. 
The  activity  of  the  amboceptor  is  far  less  subject  to 
external  influences  which  bring  about  its  deteriora- 
tion, and  it  is  therefore  extremely  rare  to  find  that 
the  imperfection  in  the  reaction  arises  from  this 
source. 

In  testing  several  specimens  of  serum  at  one 
time  it  happens  occasionally  that  some  specimens 
are  slower  in  completing  the  haemolytic  reaction 
than  others.  The  cause  of  this  slowness  is  not  pres- 
ent in  the  complement  or  amboceptor,  but  in  the 
specimens  themselves.  In  such  cases  the  specimens 
are  found  to  contain  anticomplementary  substances 
which  react  with  and  reduce  the  activity  of  the  com- 
plement. To  remove  this  source  of  error,  it  is  neces- 
sary to  heat  the  serum  to  55°  C.  for  twenty  minutes 
and  use  four  drops  for  the  test.  The  difficulty  may 
be  obviated  in  some  cases  by  collecting  specimens  of 
serum  to  be  tested  just  before  meal-time,  because  the 
anticomplementary  substance  is  closely  associated 
with  the  absorption  of  the  chyle  into  the  circulation 
soon  after  the  meal. 

3.  The  quality  and  quantity  of  the  antigen  can 
also  be  sources  of  error.  If  one  uses  poor  antigen, 
either  there  will  be  no  positive  reaction  at  all,  or 
weak  positive  reactions  will  be  entirely  overlooked. 


SERUM  DIAGNOSIS  OF  SYPHILIS.  93 

If,  on  the  other  hand,  an  excessive  amount  of  unfrac- 
tionated  crude  antigen  is  employed  certain  nonspecific 
weak  reactions  may  become  manifest,  or  a  false  posi- 
tive reaction  even  may  be  obtained,  as  the  result 
of  the  action  of  anticomplementary  substances 
sometimes  contained  in  preparations  of  the  antigen. 
These  sources  of  error  can  be  entirely  excluded  by 
choosing  an  antigen  that  has  been  carefully  prepared 
and  standardized  by  an  experienced  serologist,  which 
is,  indeed,  one  of  the  advantages  which  the  employ- 
ment of  my  system  offers. 

Apart  from  these  suggestions,  which  are  essential 
in  order  to  obtain  reliable  results  with  the  present 
system,  a  few  words  may  be  added  concerning  the 
making  of  the  reactions  on  a  larger  scale.  In  other 
words,  if  it  is  desired  to  make  the  test  with  larger 
quantities,  one  has  simply  to  multiply  the  quantity 
of  each  factor  employed.  Thus  one  may  use  0.1  c.c. 
of  the  complement,  0.05  c.c.  of  the  patient's  serum, 
1  c.c.  of  a  5  per  cent,  suspension  of  the  washed  human 
corpuscles,  and  2  units  of  the  amboceptor  (titrated 
with  the  above  complement  and  corpuscle-suspension 
unit),  in  a  total  volume  of  5  c.c.  of  physiological  salt 
solution.  This  increase  in  the  relative  quantities  of 
each  constituent  offers  one  advantage  and  one  dis- 
advantage. The  advantage  is  that  the  intensity  of 
the  reaction  can  be  more  minutely  measured  through 
the  liberation  of  the  heemoglobin,  as  the  number  of 


94  SERUM  DIAGNOSIS  OF  SYPHILIS. 

red  corpuscles  is,  of  course,  much  larger.  The  dis- 
advantage arises  from  the  unnecessary  waste  of  ma- 
terial. For  the  worker  in  a  regularly  equipped  bio- 
logical laboratory  this  waste  may  make  but  little  dif- 
ference, but  for  those  who  intend  to  do  the  test  in 
a  private  laboratory  the  exercise  of  economy  is  highly 
desirable. 

When  the  serum  to  be  tested  has  previously  been 
inactivated  by  being  heated  to  56°  C,  the  amount  of 
serum  used  must  be  from  four  to  five  times  greater 
than  that  prescribed  for  the  fresh  or  unheated  serum, 
since  one  effect  of  the  inactivation  is  to  reduce  the 
content  of  the  antibody  to  about  one-fourth  or  one- 
fifth  of  the  original  strength. 

In  the  following  chapter  the  writer  will  point 
out  the  effect  of  inactivation  upon  the  antibody  con- 
tent of  serum.  This  has  bearing  not  only  on  what 
has  been  said  above,  but  with  equal  if  not  greater 
directness  upon  the  original  Wassermann  and  other 
systems  requiring  inactivation  of  the  patient's  serum. 


IX. 

INACTIVATION  OF  THE  SERUM  IN  RELATION  TO  THE 
SYPHILIS  REACTION. 

It  will  be  recalled  that  the  serum  of  patients  to 
be  tested  is  employed  either  in  the  fresh  state  or  after 
inactivation  at  56° C.  According  to  the  method  used 
in  the  systems  of  Wassermann,  Detre,  Bauer,  Boas, 
and  Browning,  the  serum  is  previously  heated  to 
56°  C.  for  half  an  hour,  in  order  to  destroy  all 
the  native  complement  present  in  it.  On  the  other 
hand,  in  the  systems  of  Hecht,  Stern,  and  Tscher- 
nogubow  the  serum  is  emploj^ed  in  the  fresh  state, 
since  in  these  systems  the  native  complement  is 
utilized.  Unlike  these  two  sets  of  systems,  the 
one  which  I  offer  enables  one.  to  use  either  fresh,  or 
old,  or  inactivated  serum,  the  only  difference  being 
that  when  the  serum  has  been  inactivated  a  somewhat 
larger  quantity  of  it  must  be  employed. 

We  will  now  consider  the  reasons  why  one  set  of 
workers  employ  for  the  reaction  the  inactivated,  and 
another  the  fresh  serum.  Wassermann,  Bauer,  and 
others  inactivate  the  serum  simply  to  destroy  the 
native  complement,  which  varies  in  different  speci- 
mens of  serum,  and  in  order  to  substitute  this  un- 

95 


96  SERUM  DIAGNOSIS  OF  SYPHILIS. 

known  content  by  a  uniform  amount  of  guinea-pig 
complement  of  known  activity.  Hecht  and  Stern, 
however,  found  that  when  the  test  is  made  with  fresh 
serum,  so  as  to  employ  the  native  complement,  the 
reaction  is  more  sensitive  than  when  the  inactivated 
serum  is  used.  On  what  does  this  greater  delicacy  of 
reaction  of  the  fresh  serum  depend?  Since  this  point 
has  not  been  touched  upon,  I  have  made  a  careful 
study  of  it,  as  a  result  of  which  I  am  prepared  to 
offer  an  explanation  of  the  difference  existing 
between  fresh  and  inactivated  serums. 

The  first  question  which  I  asked  myself  was :  Is  the 
so-called  syphilitic  antibody  affected  in  the  process 
of  inactivation?  It  had  previously  been  found  that 
this  antibody  is  completely  destroyed  at  temperatures 
between  72°  and  80°  C.  in  about  twenty  minutes.  I 
found  that  the  spinal  fluid  loses  its  antibody  when 
heated  to  from  75°  C.  to  80°  C.  for  twenty  minutes, 
as  had  been  previously  found  by  Marie  and  Levaditi. 
The  syphilitic  serum  I  observed  to  have  become  in- 
active at  72°  C,  but  the  coagulation  of  the  protein 
interfered  in  a  high  degree  with  exact  observation. 
We  know,  therefore,  with  fair  accuracy  the  limit  of 
temperature  at  which  the  total  destruction  of  the  anti- 
body is  established,  but  we  know  almost  nothing  of 
the  rate  of  destruction  which  takes  place  at  lower 
temperatures.    Sachs  states  that  the  antibody  content 


SERUM  DIAGNOSIS  OF  SYPHILIS. 


97 


of  syphilitic  serum  is  considerably  reduced  by  tem- 
peratures of  60°  C.  I  have  found,  on  subjecting  a 
specimen  of  serum  from  a  case  of  untreated  secondary 
syphilis  to  temperatures  of  45°,  50°,  55°,  and  60°  C. 
for  twenty  minutes  and  then  determining,  by  fixation 
tests,  the  amount  of  the  antibody  available,  that  the 


50c 


55  c 


Curve  1.— Heating  of  a  syphilitic  serum  to  different  temperatures  for  20  minutes  (in  a 

water-bath) . 

syphiHtic  antibody  is  greatly  reduced  even  at  45°.  At 
50°  C.  it  is  reduced  to  about  one-half,  at  55°  C.  to 
about  one-fourth,  etc.,  as  is  shown  in  Curve  1.  I 
next  studied  the  rate  of  destruction  of  the  antibody 
at  the  temperature  of  55°  C,  at  five,  ten,  twenty, 
thirty,  and  sixty  minute  periods.  The  results  were 
rather  unexpected,  since  the  rate  of  destruction  is 
greatest  during  the  first  five  minutes,  during  which 
time  the  antibody  strength  is  reduced  about  one-third 


98  SERUM  DIAGNOSIS  OF  SYPHILIS. 

of  the  original.  After  thirty  minutes  it  has  been 
reduced  one-fourth  to  one-fifth,  and  at  the  end  of 
one  hour  to  about  one-tenth  of  the  original,  as  can 
be  seen  by  reference  to  Curve  2.  In  studying  the 
serum  from  a  case  of  leprosy,  I  found  that  the  diminu- 
tion of  the  antibody  strength  went  on  in  precisely 


CUBVE  2.— Heating  of  three  different  samples  of  syphilitic  sera  to  55°  C.  for  varying 
lengths  of  time  (in  a  water-bath). 

the  same  way  as  in  the  specimens  obtained  from 
syphilis  (Curve  3). 

It  has  been  stated  by  certain  investigators  that  the 
fresh  serum  of  nonsyphilitic  cases — as,  for  example, 
cases  of  carcinoma — may  give  a  positive  reaction, 
and  that  this  reaction  disappears  when  such  a  serum 
has  been  previously  heated  to  56°  C.  for  thirty 
minutes,  and  that  therefore  the  two  groups  of  positive 
reaction,  specific  and  nonspecific,  can  be  distinguished 
by  the  employment  of  inactivated  serum.     I   have 


SERUM  DIAGNOSIS  OF  SYPHILIS. 


99 


not  been  able  to  find  any  very  accurate  studies  of 
this  topic,  which  on  the  whole  is  so  very  important. 
My  recent  studies  on  the  Bordet-Gengou  fixation 
phenomena  in  general  revealed  an  unexpected  fact, 
namely^  that  in  the  majority  of  active  human  sera 
irrespective  of  sources  there  exists  a  constituent  which 


0        5m       j'QTn  2'0''"  JO'' 

CUKVE  3. — Heating  of  a  serum  from  a  case  of  leprosy  to  55°  C. 

fixes  complement  when  mixed  with  certain  proteins 
such  as  nucleo proteins^  pepton,  alhumoses,  and  many 
other  autolytic  decomposition  products  of  proteins. 
I  designated  this  phenomenon  proteotropic  fixation 
in  contradistinction  to  lipotropic  fixation  due  to  the 
action  of  certain  lipoids  upon  the  syphilitic  serum. 
This  proteotropic  fixation  is  of  course  nonspecific^  but 
difficult  to  differentiate  from  the  real  specific  Bordet- 
Gengou  as  well  as  the  syphilis  reactions.  Fortunately 


100  SERUM  DIAGNOSIS  OF  SYPHILIS. 

this  proteotropic  reaction  does  not  occur  when  the 
serum  is  previously  heated  to  55°  C.  for  twenty  min- 
uteSj  while  the  lipotropic  fixation  is  not  thereby  abol- 
ished.    ThuSj  no  active  human  serum  should  be  em- 
ployed for  fixation  test  when  these  proteins  can  not  be 
excluded  from  the  extracts  serving  as  antigens.    Con- 
sidering the  syphilis  reaction  in  the  light  of  this 
revelation  it  becomes  at  once  evident  that  none  but  the 
inactivated  sera  should  be  used  for  the  test  when 
aqueous  or  alcoholic  extracts  of  macerated  organs 
are  employed  as  antigens,  because  these  antigen  prep- 
arations contain  various  proteids  and  are  liable  to 
give  a  nonspecific  proteotropic  fixation  with  active 
sera.    On  the  other  hand,  there  is  no  such  a  danger  in 
using  active  sera  when  one  employs  pure  lipoidal 
substances  as  antigen.     This  is  the  reason  why  I 
recommend  the  use  of  aceton-insoluble  tissue  lipoids 
for  antigen  and  the  experiences  of  many  investigators 
on  over  20,000  cases  prove  that  my  claim  is  correct. 
Thus  the  introduction  of  pure  lipoidal  antigen  pre- 
pared by  my  method  rendered  it  for  the  first  time  pos- 
sible to  employ  an  active  human  serum  for  the  diag- 
nosis of  syphilis.    One  can  easily  see  how  erroneous 
it  would  be  to  employ  certain  other  preparations  of 
antigen  containing  various  proteids  in  my  method  in 
which  active  sera  are  principally  used.    In  fact,  one 
or  two  investigators  committed  this  error  and  placed 


SERUM  DIAGNOSIS  OF  SYPHILIS.  101 

the  blame  on  the  system.  The  results  obtained  by 
using  active  human  serum,  and  the  lipoids  just  re- 
ferred to  are  comparable  to  those  obtained  by  inacti- 
vated serum  and  any  other  suitable  syphilitic  antigens 
and  are  perfectly  specific} 

^  It  is  an  erroneous  conception  that  my  system  uses  only  active 
serum.  On  the  contrary,  it  can  use  an  old  or  inactivated  serum  as 
well.  Whether  the  active,  old,  or  inactivated  serum  is  used  is  only  a 
matter  of  personal  choice,  provided  that  the  rules  I  prescribed  for  active 
and  inactive  sera  are  observed. 


X. 

TECHNIC  OF  THE  WASSERMANN  SYSTEM. 

In  performing  the  test  by  the  original  Wasser- 
mann  system  five  different  factors  are  required,  viz.: 
antigen,  patient's  serum,  complement,  amboceptor, 
and  blood-corpuscles.  The  source  and  mode  of 
preparation  of  these  factors  will  be  given  in  detail 
below. 

PREPARATION    OF    ANTIGEN. 
A.    AQUEOUS  EXTRACTS. 

1.  Wassermann's  Method. — The  liver  or  spleen 
of  a  congenitally  syphilitic  foetus  is  preferred.  Take 
the  organ  and  cut  it  up  into  very  small  pieces  with 
a  pair  of  scissors  and  mix  the  tissue  with  four  parts 
of  physiological  salt  solution  to  which  phenol  in  the 
proportion  of  0.5  per  cent,  is  added.    For  example: 

S60.0  c.c.  salt  solution   (0.85  per  cent.) 
100.0  grams  liver 
40.0  c.c.  phenol    (5  per  cent.) 

This  mixture  is  thoroughly  shaken  in  a  dark  bottle 
for  twenty-four  hours  by  means  of  a  shaking  machine. 
The  tissue  pieces  are  separated  by  centrifugalization 
and  the  brownish,  opalescent  supernatant  fluid  is  used 
for  antigen.  It  should  be  preserved  in  a  rubber- 
stoppered   dark   flask   in   the   refrigerator.      Upon 

102 


SERUM  DIAGNOSIS  OF  SYPHILIS.  103 

standing  a  precipitate  falls  to  the  bottom  of  the  con- 
tainer. This  precipitate  should  not  be  used.  As  much 
of  the  clear  supernatant  fluid  as  is  necessary  for  the 
day's  work  should  be  poured  off,  and  the  remainder 
put  back  on  ice  immediately. 

As  to  the  stability  of  this  extract,  there  is  no 
agreement  among  investigators,  whose  experiences 
differ  widely  upon  this  question.  Wassermann, 
Neisser,  Bruck  and  Schucht  found  that  it  is  very 
unstable,  soon  becoming  too  anticomplementary  for 
use.  Citron  once  prepared  a  watery  solution  of  anti- 
gen which  he  divided  into  three  portions.  He  kept 
one  part  for  his  own  use  and  the  other  two  he  sent 
to  other  laboratories.  One  of  these  reported  to  him 
after  the  lapse  of  a  week  that  the  antigen  had  become 
inactive;  the  other  sent  a  similar  report  after  four 
weeks.  The  portion  which  he  kept  was  unaltered 
three  months  after  its  preparation.  So  that  it  would 
seem  the  stabiHty  of  the  antigen  depends  greatly 
upon  the  mode  of  its  preservation.  My  own  experi- 
ence shows  that  the  liver  of  every  congenitally 
syphilitic  fcetus  does  not  always  yield  a  good  antigen 
and  that  when  once  prepared  in  the  above  manner  it 
may  deteriorate  within  a  few  weeks. 

For  the  extract  used  in  the  control  tests  a  normal 
organ  should  be  similarly  prepared. 

2.  Marie  and  Levaditi's  Method. — Mash  the  liver 
of  a  congenitally  syphilitic  foetus  and  dry  in  a  vacuum 


104  SERUM  DIAGNOSIS  OF  SYPHILIS. 

and  then  pulverize  it.  The  powder  is  suspended  in 
four  parts  of  physiological  salt  solution  and  the  mix- 
ture centrifugalized  after  twenty-four  hours'  extrac- 
tion.    The  clear  supernatant  fluid  is  used. 

3.  Morgenroth  and  Stertz's  Method. — Preserve 
the  organ  (syphilitic  liver)  in  frozen  state  {in  Frigo) , 
and  cut  off  a  small  piece  each  time  for  use  in  the  test. 
Mash  this  bit  of  tissue  with  sea-sand  and  extract  it 
with  four  parts  of  physiological  salt  solution.  Filter 
through  paper,  and  use  the  filtrate. 

The  most  important  point  concerning  antigen  is 
to  employ  the  proper  quantity  in  the  test.  It  has  been 
made  a  general  rule  that  that  dose  of  antigen  must 
be  selected  which  does  not  bind  complement  even 
when  the  antigen  is  used  in  double  quantity.  The 
usual  aqueous  preparation  may  be  used  in  0.1  c.c.  or 
0.2  c.c.  doses. 

B.    ALCOHOLIC    EXTRACTS. 

1.  Forges  and  Meier's  Method. — Cut  up  a  normal 
or  syphilitic  liver  into  small  pieces,  extract  with  five 
volumes  of  absolute  alcohol  for  twenty-four  hours, 
and  filter  through  coarse  filter-paper.  The  filtrate  is 
evaporated  in  a  vacuum  at  a  temperature  below  40°  C. 
The  sticky  mass  resulting  is  then  used  to  prepare  a  1 
per  cent,  suspension  in  physiological  salt  solution  with 
the  addition  of  0.5  per  cent,  of  phenol.  This  emulsion 
is  well  shaken  and  filtered  through  fine  paper.  The 
minimal  dose  which  shows  inhibition  of  haemolysis  is 


SERUM  DIAGNOSIS  OF  SYPHILIS.  105 

determined  by  titration,  and  half  of  this  amount  is 
used  for  the  test.  With  a  strong  syphilitic  serum 
0.025  c.c.  may  give  a  complete  reaction,  but  0.2  or 
0.3  c.c.  is  usually  necessary  for  the  test.  The  authors 
found  a  preparation  of  lecithin  (Kahlbaum)  to  be 
equivalent  in  antigenic  property  to  the  alcoholic  ex- 
tract, but  later  investigators  have  found  such  a  prepa- 
ration unreliable  as  an  antigen. 

2.  Landsteiner,  3Iuller  and  PotzVs  Method. — 
Extract  mashed  guinea-pig's  heart  or  liver  with  alco- 
hol for  about  ten  to  twelve  hours  at  60°  C,  in  the 
ratio  of  one  gram  of  tissue  to  50  c.c.  of  95  per 
cent,  alcohol.  Filter  through  paper  and  preserve  the 
filtrate  at  room  temperature.  Use  two  drops  of  the 
solution  for  the  test. 

3.  Michaelis  and  JLesser's  Method. — Shake 
minced  normal  or  syphilitic  liver  with  ten  volumes  of 
absolute  alcohol  for  ten  to  twelve  hours.  Use  glass 
beads  to  facilitate  thorough  extraction.  After  twenty- 
four  hours  the  clear  supernatant  portion  is  poured  or 
pipetted  off  and  used  as  antigen.  Every  time  the 
test  is  to  be  made  one  part  of  this  extract  is  mixed 
with  four  parts  of  physiological  salt  solution  and  1 
c.c.  of  this  emulsion  is  used.  The  emulsion  becomes 
milky  and  tends  to  form  a  precipitate  on  standing 
and  should  be  thoroughly  shaken  before  using.  Re- 
cently Michaelis  has  advocated  the  use  of  an  alcoholic 
extract  of  normal  human  heart. 


106  SERUM  DIAGNOSIS  OF  SYPHILIS. 

4.  The  Writer's  Method. — Extract  minced  tissue 
of  syphilitic  or  normal  heart,  liver  or  kidney  (man, 
beef,  sheep,  etc. )  with  ten  volumes  of  95  per  cent,  alco- 
hol for  about  six  or  seven  days  at  S7°  C.  Filter 
through  paper  and  evaporate  the  filtrate  by  means  of 
a  fan  at  a  temperature  below  40°  C.  The  resinous 
residue  which  results  should  be  extracted  with  ether, 
and  this  ethereal  solution  then  allowed  to  evaporate  to 
dryness  in  the  air.  Take  up  the  residue  of  this  ethe- 
real extract  with  a  small  quantity  of  ether  and  frac- 
tionate with  ten  volumes  of  aceton.  Separate  the 
sticky  precipitate  by  pouring  off  the  supernatant  ace- 
ton carefully,  allow  the  remainder  of  the  aceton  to 
evaporate,  and  preserve  the  resinous  mass  in  an  air- 
tight jar.  For  the  test  make  a  0.3  per  cent,  solution 
in  physiological  salt  solution  by  first  dissolving  the 
resinous  mass  in  a  small  quantity  of  ether  and  then 
mixing  it  with  the  salt  solution.  Each  preparation 
should  be  tested  before  using  to  determine  its  re- 
liability and  dosage.^  Usually  0.1  c.c.  to  0.2  c.c.  is 
suitable.    Kept  on  ice  the  emulsion  is  fairly  stable. 

C.    ARTIFICIAL.  ANTIGEN. 

Sachs  a7id  Rondoni  advise  the  following  as  a 
suitable  antigen  for  use  in  the  Wassermann  reaction : 

^  The  aqueous  as  well  as  the  alcoholic  extracts  prepared  by  any 
method  should  be  similarly  tested  before  use. 


SERUM  DIAGNOSIS  OF  SYPHILIS.  107 

Mixture  A.  Mixture  B. 

Sodium   oleate    (Kahlbaum)    2.5  1.0 

Lecithin  (Ovo-Merck) 2.5  1.0 

Oleic    acid    (Kahlbaum)     0.75  1.5 

Distilled  water   12.5  5.0 

Alcohol  ad 1000.0  ad  1000.0 

The  authors  advise  the  use  of  either  of  these 
formulse  in  a  dilution  of  1  part  of  the  above  to  five 
of  physiological  salt  solution,  the  reagent  being  thor- 
oughly mixed  with  the  salt  solution.  No  precipitate 
should  be  formed.  The  test  doses  advised  for  each 
serum  to  be  examined  are:  0.15,  0.25,  0.4  c.c. 

Schilrmann's  Method. — This  investigator  recently 
published  this  formula  as  an  antigen:  Lecithin,  0.30 
gram  in  50  c.c.  absolute  alcohol ;  sodium  glycerophos- 
phate, 0.3  gram  in  5  c.c.  physiological  salt  solution. 
Thirty  c.c.  of  the  above  are  mixed  with  5  c.c.  lactic 
acid  and  10  c.c.  of  ammonium  vanadinate    (1   per 

cent. ) . 

patient's  serum. 

To  get  serum  for  the  Wassermann  test  blood  is 
drawn  from  a  vein  and  the  serum  which  separates  after 
clotting  is  inactivated  at  56°  C.  for  half  an  hour, 
preferably  within  twent}^-four  hours  after  its  with- 
drawal from  the  patient.  Cerebrospinal  fluid  should 
be  used  without  inactivation.  The  test  doses  are  0.1 
c.c.  and  0.2  c.c. 

COMPLEMENT. 

One  cubic  centimetre  of  guinea-pig's  serum  in 
1 :10  dilution  is  used.    The  serum  should  not  be  older 


108  SERUM  DIAGNOSIS  OF  SYPHILIS. 

than  forty-eight  hours,  and  it  should  be  carefully 
preserved  in  the  ice  chamber  when  not  in  use. 

AMBOCEPTOR. 

The  amboceptor  for  the  Wassermann  test  is  pro- 
duced by  immunizing  rabbits  against  sheep-corpuscles. 
The  writer  has  been  very  successful  in  obtaining  an 
amboceptor  of  high  titre  by  using  successive  injec- 
tions of  washed  corpuscles  in  doses  of  two,  four, 
eight,  and  twelve  cubic  centimetres  at  intervals  of 
four  or  five  days,  and  bleeding  the  animal  nine  or 
ten  days  after  the  last  injection.  The  corpuscles 
should  be  centrifugalized  at  least  twice  with  a  large 
quantit}^  of  physiological  salt  solution  and  the  original 
bulk  of  the  defibrinated  blood,  which  had  been  marked 
before  centrifugahzation,  restored  by  the  addition  of 
salt  solution.  The  injections  should  be  made  intra- 
peritoneally. 

One  unit  of  amboceptor  should  be  determined 
by  titrating  against  1  c.c.  of  a  5  per  cent,  suspension 
of  washed  sheep-corpuscles,  using  0.5  c.c.  of  1 :  10 
dilution  guinea-pig  complement.  Two  units  are  used 
in  the  test. 

CORPUSCLE    SUSPENSION. 

One  cubic  centimetre  of  a  5  per  cent,  suspension 
of  washed  sheep's  corpuscles  is  used.  The  blood 
should  be  fresh,  not  older  than  three  days,  and  should 
be  kept  on  ice  when  not  in  use. 


SERUM  DIAGNOSIS  OF  SYPHILIS.  109 

METHOD    OF    APPLYING    THE    TEST. 

In  applying  the  test  according  to  the  original 
Wassermann  method  with  a  watery  antigenic  extract, 
the  investigator  should  use  as  many  test-tubes  as  are 
indicated  in  Table  2. 

Put  the  required  amounts  of  serum,  complement, 
and  antigen  into  the  respective  tubes,  and  bring  the 
total  quantity  of  the  mixture  up  to  3  c.c.  by  the  addi- 
tion of  salt  solution.  Mix  the  contents  of  the  tubes 
well  and  incubate  in  the  thermostat  for  one  hour  at 
37°  C.  At  the  end  of  this  period  add  to  everj^  tube 
amboceptor  and  corpuscle  suspension  in  the  quantity 
prescribed  above,  mix  weU,  and  incubate  again  for 
two  hours.  Then  remove  the  tubes  to  an  ice-chest  for 
twenty  hours,  when  the  test  is  complete  and  the  re- 
sults are  ready  for  reading. 

If  the  test  has  been  properly  carried  out,  there  will 
be  complete  haemolysis  in  every  control  tube  excepting 
in  the  tube  containing  syphilitic  serum  and  syphilitic 
antigen  (positive  control,  vide  Table  2) .  If  the  control 
tubes  are  correct,  the  tubes  containing  the  sera  to  be 
examined  can  be  read  for  the  final  result.  In  this 
series  all  the  tubes  containing  other  than  a  syphilitic 
organ  extract  should  be  completely  hgemolysed. 

In  the  tubes  containing  the  unknown  serum  and 
a  syphilitic  organ  extract,  there  may  or  may  not  be 
haemolysis  according  as  the  serum  contains  syphilitic 


110  SERUM  DIAGNOSIS  OF  SYPHILIS. 

antibodies  or  not.  In  the  former  event  there  wiU  be 
inhibition  of  heemolysis,  either  total  or  partial;  in  the 
latter,  the  tubes  should  be  completely  hsemolysed. 

The  degree  of  inhibition  of  haemolysis  varies  ac- 
cording to  the  amount  of  syphilitic  antibody  present; 
if  this  is  large  in  amount,  or,  in  other  words,  if  com- 
plete inhibition  occurs  in  the  tube  containing  0.1  c.c. 
serum  and  0.1  c.c.  antigen,  the  result  can  be  graphi- 
cally represented,  according  to  Citron,  thus:  H — I — I — h. 
If  inhibition  of  hasmolysis  is  incomplete  in  the  tube 
containing  0.1  c.c.  of  serum  but  complete  in  that  con- 
taining 0.2  c.c,  the  result  is  expressed  thus:  H — 1 — [-. 
These  reactions  are  usually  called  strongly  positive. 
If  the  tube  containing  0.1  c.c.  serum  is  completely 
hsemolysed  while  that  containing  0.2  c.c.  shows  com- 
plete inhibition,  the  result  is  expressed  thus:  H — h. 
Incomplete  inhibition  in  the  tube  containing  0.2  c.c. 
is  expressed  thus :  +.  The  last  two  reactions  are  called 
weakly  positive.  When  inhibition  in  the  tube  con- 
taining 0.2  c.c.  is  doubtful  the  result  is  expressed 
thus :   ± . 

THE    writer's     method^    OF     PERFORMING    THE    TEST 
WITH    THE    WASSERMANN    SYSTEM. 

The  regular  Wassermann  method  can  be  greatly 
simplified  by  the  use  of  aceton-insoluble  tissue 
lipoids  as  antigen.     Other  investigators,  as  well  as 

^  This  should  not  be  confused  with  the  antihuman  system  already 
described  in  Chapter  VII. 


SERUM  DIAGNOSIS  OF  SYPHILIS. 


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112  SERUM  DIAGNOSIS  OF  SYPHILIS. 

the  writer,  have  found  that  there  is  no  essential  differ- 
ence between  the  antigen  extract  thus  prepared  and 
the  aqueous  extract,  while  the  former  in  the  hands  of 
the  writer  has  proved  much  more  stable  and  does  away 
with  the  necessity  of  using  the  normal  organ-extract 
controls,  which  render  the  original  Wassermann 
method  so  complicated  and  cumbersome. 

In  Table  3  (see  page  113)  the  exact  method  of 
performing  the  test  is  given.  It  wiU  be  noted  that 
all  the  reagents  have  been  used  in  half  the  quantity 
of  the  original  Wassermann  method.  A  uniform 
dosage  of  antigen  is  used,  which,  in  the  experience  of 
the  writer,  has  been  sufficient  to  show  the  varying 
intensity  of  the  reaction. 


SERUM  DIAGNOSIS  OF  SYPHILIS. 


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XI. 

DIAGNOSTIC  VALUE  OF  THE  SERUM  REACTION 
OF  SYPHILIS. 

The  phenomenon  of  complement-fixation  in 
syphilis  is  a  type  of  reaction  distinct  in  itself  and 
differing  widely  from  all  other  known  examples  of 
complement-fixation.  The  principal  difference  be- 
tween the  two  types  of  phenomena  arises  from  the 
nonspecific  nature  of  the  substances  that  functionate 
as  antigen  in  the  Wassermann  reaction.  We  have 
already  seen  (see  page  23)  that  the  complement  is 
absorbed  or  fixed  only  when  brought  in  contact  with 
combinations  of  specific  antigens  and  antibodies.  In 
general  it  may  be  said  that  the  specificity  of  these 
antigens  and  antibodies  can  be  compared  in  a  way 
to  the  relation  which  exists  between  locks  and  keys, 
and  it  can  be  stated  that  they  do  not  interact  with 
one  another  unless  they  are  in  exact  correspondence. 
On  the  other  hand  the  phenomenon  of  Wassermann 
is  produced  by  what  appears  to  be  a  specific  antibody 
and  certain  nonspecific  antigenic  substances.  There- 
fore in  this  case  the  law  of  specificity  does  not  operate 
in  the  same  strict  sense  as  in  other  known  examples 
of  the  Bordet-Gengou  phenomenon. 

114 


SERUM  DIAGNOSIS  OF  SYPHILIS.  116 

I  have  been  able  to  show  that  several  phosphorized 
and  non-phosphorized  lipoids  and  a  few  salts  can  act 
as  syphilitic  antigens,  and  that  there  is  no  necessary 
relation  between  them  and  a  syphilitic  infection. 
When  these  substances  are  brought  into  combination 
with  the  blood-serum  or  cerebrospinal  fluid  of  syphi- 
litic patients,  they  alter  the  fluids  in  such  a  manner 
as  to  render  them  able  to  fix  the  complement  which 
is  introduced  into  the  mixture.  It  is  this  peculiarity 
of  the  serum  of  syphilitics  upon  which  the  serum 
diagnosis  of  syphilis  is  based.  This  interesting 
property  of  syphilitic  serum  is  produced,  it  is  believed, 
by  certain  substances  existing  in  the  serum,  now  gen- 
erally designated  as  syphilitic  antibody,  although 
actually  we  are  still  entirely  ignorant  of  their  real 
nature.  There  is,  however,  little  doubt  that  they 
are  reaction  products  of  syphilitic  infection,  and  that 
they  appear  constantly  in  the  serum  of  persons  in- 
fected at  certain  stages  of  the  disease. 

For  the  purpose  of  estimating  the  value  of  the 
complement-fixation  test  as  a  clinical  method  for  the 
diagnosis  of  syphilitic  and  parasyphilitic  conditions, 
the  writer  presents  a  tabulation  of  the  results  of  in- 
vestigators.    (See  pages  116  and  117.) 

In  primary  syphilis  the  results  vary  much,  rang- 
ing from  98  per  cent.  (Detre)  to  38  per  cent. 
(Hoehne).     This  difference  may  be  accounted  for 


116 


SERUM  DIAGNOSIS  OF  SYPHILIS. 


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SERUM  DIAGNOSIS  OF  SYPHILIS. 


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118 


SERUM  DIAGNOSIS  OF  SYPHILIS. 


aside  from  technical  considerations,  by  the  state  of 
the  infection  at  the  time  of  examination.  Early  cases 
of  chancre  frequently  give  a  negative  reaction.  How- 
ever, in  this  stage  of  syphilis  the  test  is  not  usually 
necessary  excepting  in  those  cases  in  which  a  differ- 
entiation between  chancroidal  and  mixed  infection  is 
desirable,  and  in  cases  of  suspected  intra-urethral 
chancre. 

In  the  following  table  I  present  the  data  collected 
by  Craig  concerning  the  time  of  appearance  of  posi- 
tive reaction  in  primary  cases. 

Table  5a. 

Date  of  Appearance  of  Positive  Reaction  in 
Thirty-one  Cases  of  Lues. 


Days  after  initial  lesion. 

No.  of  cases  positive. 

5 

1 

8 

2 

11 

2 

13 

3 

14 

I 

17 

2 

18 

2 

19 

2 

20 

1 

21 

2 

22 

1 

23 

1 

25 

2 

28 

1 

29 

2 

30 

6 

It  will  be  seen  that  the  earliest  was  five  days  after 
the  initial  lesion  had  appeared. 


SERUM  DIAGNOSIS  OF  SYPHILIS.  119 

In  secondary  syphilis  the  highest  figures  are  those 
of  Boas  and  of  Ledermann;  the  former  got  100  per 
cent,  of  positive  reactions  in  395  cases,  the  latter  a 
similar  result  in  56  cases.  The  lowest  figures  are 
recorded  by  Hoehne,who  got  79.1  per  cent,  of  positive 
reactions  in  329  cases,  and  by  Bruck  and  Stern,  who 
examined  163  cases  with  similar  results.  The  vari- 
ations in  these  figures  cannot  well  be  accounted  for 
unless  an  analysis  of  the  stage  of  the  disease  and  the 
treatment  received  by  the  patient  at  the  time  of  doing 
the  test  are  taken  into  consideration.  The  reaction  in 
this  stage  of  syphilis  is  fairly  constant  and  a  reliable 
index  of  the  presence  of  syphilitic  antibodies  in  the 
patient's  serum. 

In  tertiary  lues  the  figures  vary  from  57.4  per 
cent.  (Bruck  and  Stern  in  47  cases)  to  100  per  cent. 
(Bruhns  and  Halberstadter  in  16  cases) .  Here  again 
the  same  uncertainty  as  to  treatment,  which  may  so 
strongly  aifect  the  reaction,  applies  as  pointed  out 
above. 

In  early  latent  cases  the  figures  vary  from  20  per 
cent.  (Bruck  and  Stern  in  50  cases)  to  85  per  cent. 
(Wassermann,  Neisser,  et  at.  in  41  cases).  By  early 
latent  cases  is  meant  those  late  secondary  cases  with- 
out symptoms.  In  late  latent  cases,  or  those  follow- 
ing the  manifest  tertiary  stage,  without  symptoms, 
the  figures  show  about  the  same  results.  The  technic 
of  the  various  investigators  and  the  reagents  used  by 


120 


SERUM  DIAGNOSIS  OF  SYPHILIS. 


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SERUM  DIAGNOSIS  OF  SYPHILIS. 


121 


them  must  be  taken  into  account  in  accepting  the 
results  of  their  work. 

General  paralysis  shows  fairly  constant  positive  re- 

Tablb  7. 


General  paralysis 

Blood-serum 

Cerebrospinal  fluid 

Number 
of  cases 

W. 
% 

N. 
% 

Number 
of  cases 

W. 

% 

N. 
% 

Noguchi 

25 

56 

11 

61 

3 

4 

2 

80 
65 
66 

86 
80 
80 
72 
66 
100 
100 

44 
5 

100 

Rosanoff  and  Wiseman. . .  . 
Corson- White 

86 
100 

Kaplan 

Kaliski 

Schradieck 

Groat 

Total 

162 

70 

73.4 

49 

100 

93 

Table  8. 


Tabes 


Blood-serum 


Number         W. 
of  cases         % 


Noguchi 

Noguchi 

Kaplan , 

Corson-White 

Kaliski 

Berghausen. . 

Fox 

Waugh 

Total. .  . 


125 

8 

205 

49 

10 

6 

3 

13 


44 
60 
70 
40 

100 


68 
72 
65 
75 
60 
66 
100 
56 


419 


62.8 


72 


actions,  ranging  from  80  per  cent,  to  100  per  cent, 
of  cases  examined.  Tabes  gives  a  somewhat  lower 
percentage,  from  40  per  cent,  to  80  per  cent.     In 


122  SERUM  DIAGNOSIS  OF  SYPHILIS. 

hereditary  syphilis  the  figures  are  fairly  constantly 
high,  the  lowest  being  87.5  per  cent.  In  cerebro- 
spinal lues  the  results  vary  from  16  per  cent,  in  12 
cases  reported  by  Hoehne  to  88.5  per  cent,  in  26 
cases  reported  by  Ledermann. 

The  writer  interpolates  the  results  obtained  by 
different  investigators  with  his  system  in  3580  cases 
of  syphilis  in  its  various  manifestations  and  stages. 
Out  of  this  total  number  1771  cases  were  examined 
by  the  Wassermann  system  at  the  same  time.  The 
results  of  comparison  show  clearly  that  my  method 
gives  a  higher  percentage  of  positive  reactions  than 
with  the  Wassermann.     (Tables  6,  7  and  8.) 

Just  how  this  difference  in  sharpness  of  reaction 
between  my  system  and  that  of  Wassermann  arises 
has  been  repeatedly  emphasized  and  there  can  be  no 
doubt  that  this  is  due  to  the  occasional  excessive  anti- 
sheep  amboceptor  present  in  some  human  sera  under 
investigation.  Kaliski  published  a  series  of  cases 
where  the  reactions  with  my  system  were  positive  and 
with  the  Wassermann  negative.  (Table  9.)  In 
Table  10  the  results  of  routine  examinations  of  the 
sera  from  different  diseases  by  the  same  investigator 
are  given. 

Table  9. — Noguchi  method  positive,  Wassermann  negative. 

N.  W. 

Congenital  syphilis +  —         Under  Hg  treatment  till  recently. 

Secondary  syphilis <+  —         Under     Hg      treatment     till     3 

months  ago. 


SERUM  DIAGNOSIS  OF  SYPHILIS. 


123 


N.  W. 

Secondary  syphilis +  <+         Under  Hg  treatment. 

Latent  syphilis <+  —         Chancre  many  years  ago. 

Tertiary  syphilis <+  — 

Tertiary  syphilis +  <+  Excess  natural  antisheep  am- 
boceptor. 

Tertiary  syphilis +  —         Gumma  pyloric  end  stomach. 

Tertiary  syphilis <+  —         Under  Hg  treatment. 

Latent  syphilis <+  —         Chronic  treatment  7  years. 

Cerebrospinal  syphilis +  — 

Cerebrospinal  syphilis +  (±)       Wassermann  almost  negative. 

Cerebrospinal  sjTjhilis +  (±  )       Wassermann  only  suspicious. 

Tabes +  —         Great  excess  amboceptor. 

Tabes +  <+         3  units  natural  amboceptor. 

Periostitis,  specific +  —         2  units  natural  amboceptor. 

Stricture  rectum +  —         Natural  amboceptor. 

Epiphysitis,  specific <+  — 

Endometritis  (abortions) +  (±)       Wassermann  suspicious. 

Osteomyelitis,  specific +  <+         No    natural     amboceptor;     goes 

negative  on  addition  of  two 
units  artificial  amboceptor. 

Hodgkin's  disease,  also  lues ....  4-  —         Chancre  about  20  years  ago. 

Proctitis,  specific <+  —         Under  Hg  treatment  till  recently 

Ulcus  criu-is,  specific +  —         Chancre  25-30  years  ago. 

+  ,  Positive.  — ,  Negative.  <+,  Weakly  positive  to  moderate.  (±),  Only 
suspicious. 

Table  10. — Routine  cases  from  the  wards  for  diagnosis  with  Wasser- 
mann and  Noguchi  systems  {Kaliski). 

Neg.  Pos. 

Anemias  —  splenic     pernicious, 

secondary 8  0 

Chronic  endocarditis 26  0 

Chronic  endocarditis  and  nephri- 
tis   5  1 

Nephritis,  acute  and  chronic.  ...  21  1            Chancre  three  years  ago. 

Myocarditis 5  1             Autopsy. 

Hodgkin's  disease 8  1            Chancre  twenty  years  ago. 

Leukemias 4  0 

Banti's  disease 4  0 

Aortic  regurgitation,  aortitis.  ..  .  3  1 

Aortic  stenosis 2  0 

Aneurism 2  1 

Arteriosclerosis 6  2 

Paroxysmal  tachycardia 1  0            Perforated  septum,  tertiary. 

Arteriosclerosis 6  2 

Heart-block 1  0 

Cirrhosis  liver 4  6 

Chronic  cholecystitis 3  0 

Cholelithiasis 4  0 

Acute  yellow  atrophy 1  0 

Arthritis  deformans 8  5 

Arthritic  conditions — chronic  in- 
fectious, Still's  metaboHc,  etc..  25  0 


124 


SERUM  DIAGNOSIS  OF  SYPHILIS. 


Auto-intoxication,  Brill's  disease, 

etc 

Typhoid 

Dysentery 

Gastric  conditions — gastralgia, 
hyperacidity,  gastritis,  ulcer.  . 

Pulmonary  conditions — pneumo- 
nia, abscess,  asthma,  emphy- 
sema, pleurisies,  timior 

Pulmonary  tuberciilosis 

Diabetes  mellitus 

Diabetes  insipidus 

Asthenia 

Achondroplasia 

Hydrocephalus 

Rickets 

Marasmus 

Craniotabes 

Varicella 

Plumbism 

Myxedema 

Gout 

Trichinosis 


Neg.      Pes. 


Chancre     twenty-eight     months 


Gumma     pylorus    at    operation. 
See  Table  2. 


15 


10 


208 


0 
0 
0 
1 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 

22 


Debmatological  Conditions 


Ulcus  cruris 

Eczema  seborrheal. . . 

Tuberculosis  cutis 

Leukoplakia  buccaUs. 

Lichen  planus 

Sycosis 

Scleroderma , 


Leprosy 

Sporotrichosis 

Lupus  erythematosus 

Psoriasis 

Herpes  zoster  and  progenitalis . 
Dermatitis 


Cerebral  thrombosis;   hemiplegia 

Cerebral  endarteritis 

Myelitis 

Sciatica 

Peripheral  neuritis 

Trigeminal  neuralgia 

Myalgia 

Neuralgia 

Neurasthenia  and  hysteria 


5 

3 

Three  specific,  five  varicose  ulcers. 

2 

0 

1 

0 

4 

0 

No  history  syphilis  in  any. 

2 

0 

2 

0 

1 

1 

Wassermann   and   Noguchi  both 
weakly    positive.     No    history 

0 

3 

of  syphilis. 

1 

0 

1 

0 

2 

0 

5 

0 

2 

0 

CAL 

Conditions 

11 

1 

3 

1 

6 

2 

In  two  cases  diag.  =  specific. 

2 

0 

1 

0 

2 

0 

2 

0 

5 

0 

15 

0 

SERUM  DIAGNOSIS  OF  SYPHILIS. 


125 


Neg.  Pos, 

Cerebral  and  cerebellar  tumor ...  6  0 

Abscess  brain 2  0 

Cerebellar  ataxia 2  0 

Tumor  cord  and  spine 3  0 

Spastic  and  ataxic  paraplegia ...  4  0 

Specific  paraplegia 1  2 

Optic  neuritis 2  0 

Chorea 2  0 

Pachymeningitis 2  0 

Serous  and  tubercular  meningitis  2  0 

Meningoencephalitis 0  2 

Myasthenia  gravis 1  0 

Epilepsy 2  0 

Multiple  sclerosis 3  0 

Mongolian  idiocy,  imbecihty. ...  4  0 

Friedreich's  ataxia 1  0 

Progressive  muscular  atrophy ...  2  0 

Tabes 3  9 

Paresis 2  3 

Cerebrospinal  syphilis 3  12 

Paralysis  agitans 3  0 

SuRGicAx  Conditions 

Carcinomata  and  sarcomata 38  1 

Tuberculous    conditions    (bones, 

glands,  testis,  peritoneum,  etc.)  17  0 
Bone  conditions  (osteoperiostitis, 

pain,    abscess,     osteomyehtis, 

multiple  exostosis,  etc.) 17  0 


Rectal  conditions  (stricture,  fis- 
tula in  ano,  proctitis,  abscess) . 

Thrombo-angiitis  obliterans .... 

Gynecologic  conditions  (endome- 
tritis, ectopics,  fibroids,  abor- 
tions)   


Genito-urinary  conditions  (stric- 
ture, cystitis,  hernia,  trabecu- 
lar and  atonic  bladder,  hema- 
toma and  tumor  of  testis,  ren 
mobiUs,  enlarged  prostate, 
undescended  testicle,  hydro- 
cele, calculus,  spermatocele, 
impotence,  orchitis) 

Eye  conditions  (keratitis,  iritis, 
cyclitis,  corneal  opacities,  trau- 
ma, ophthalmoplegia) 

Cerebral  thrombosis;  hemiplegia 
Cerebral  endarteritis 


2  2 

30  0 


6  8 


Spinal  fluid  positive  in  one  case. 


Spinal  fluid  positive  in  both. 


One  case  of  suspected  paresis. 


Chancre  years  ago  in  one  case. 


Positive  conditions  confirmed  by 
histological  examination  and 
subsequent  course. 

Both  positive  cases  tertiary  lues. 


Eight  out  of  twelve  women  with 
frequent  abortions  and  still- 
births give  positive  reactions. 


28 


10 


11 
3 


Both  orchitis  cases  positive. 


Two    cases    interstitial    keratitis 
positive. 


126  SERUM  DIAGNOSIS  OF  SYPHILIS. 

Neg.  Pos. 

Myelitis 6  2  In  two  cases  diag.  =  specific. 

Sciatica. 2  0 

Peripheral  neuritis 1  0 

Trigeminal  neuralgia 2  0 

Myalgia 2  0 

Neuralgia 5  0 

Neurasthenia  and  hysteria 15  0 

Cerebral  and  cerebellar  tumor. . .  6  0 

Abscess  brain 2  0 

Cerebellar  ataxia 2  0 

In  the  early  period  after  the  introduction  of  my 
system  the  superior  delicacy  of  the  reaction  aroused 
a  suspicion  among  a  few  clinicians  that  it  might  give 
positive  reaction  in  nonsyphilitic  cases.  That  their 
fear  was  wholly  ungrounded  will  be  shown  from  the 
fact  that  none  but  an  unskilled  and  hasty  serologist 
obtained  a  positive  reaction  in  ordinary  nonsyphilitic 
cases,  and  such  results  are  no  longer  obtained  by  any 
other  workers.  It  may  be  well,  however,  to  record 
here  that  the  following  investigators  did  not  get  posi- 
tive reactions  in  nonsyphilitic  cases.*      (Table  11.) 

Table  11. 

Noguchi 1642 

Kaliski 750 

Jeffries  and  Pease 300 

Schwartz  (B.) 250 

Robinson  (Orleman) 250 

Lederer 150 

Fox 113 

Groat 51 

Corson-White 183 

Craig 214 

Potter  (Alfred)         45 

Schradieck 1500 

Total 5448 

*  Cases  of  leprosy,  malaria,  yaws  or  certain  cachectic  conditions 
where  the  Wassermann  sj^stem  is  also  positive  are  not  included  here. 


SERUM  DIAGNOSIS  OF  SYPHILIS. 


127 


Orleman-Robinson  has  made  an  extensive  study 
expressly  for  the  purpose  of  determining  whether  or 
not  the  use  of  active  serum  in  my  system  gives  an 
occasional  positive  reaction  in  nonsyphilitic  derma- 
tological  conditions.  Her  results  with  236  cases  were 
uniformly  negative.  (Table  12.)  For  controls  180 
cases  of  syphilis  were  also  examined,  with  the  results 
quoted  elsewhere  in  this  book. 


Table 

Acne  vulgaris 18 

Acne  rosacea 6 

Alopecia  areata 10 

Dermatitis  herpetiformis 3 

Dermatitis  traumatica 6 

Dermatitis  venenata 4 

Eczema 25 

Epithelioma 30 

Erythema  multiforme 5 

Erysipelas 3 

Erysipeloid 3 

Favus 2 

Scabies 6 

Herpes  zoster 10 

Hydrocystoma 3 


12. 

Ichthyosis  4 

Impetigo  contagiosa 14 

Lichen  planus  .• 10 

Lichen  rubra  pilaris 1 

Lupus  erythematosus 10 

Lupus  vulgaris 4 

Molluscum  contagiosum 3 

Naevus  pigmentosus 3 

Pityriasis  rosea 4 

Pityriasis  versicolor 3 

Psoriasis 15 

Purpura 5 

Trichophytosis 10 

Tuberculosis  cutis ,  .  3 

Urticaria 13 


The  results  of  the  analysis  of  the  cerebrospinal 
fluid  in  general  paralysis  vary  from  73  per  cent. 
(Marie  and  Levaditi,  and  Noguchi  and  Moore)  to 
100  per  cent.  (Morgenroth  and  Stertz) .  The  results 
are  uniformly  high,  especiallj^  when  contrasted  with 
tabes  and  cerebrospinal  lues  ( Table  13) .  In  tabes  the 
figures  vary  from  54.5  per  cent,  to  66.6  per  cent.  In 
cerebral  syphilis  the  presence  of  the  binding  substance 


128 


SERUM  DIAGNOSIS  OF  SYPHILIS. 


is  very  uncertain;  Plant  examined  four  cases  with 
uniformly  negative  results,  while  Henderson  obtained 
positive  reactions  in  most  of  his  cases. 

Table  13. — Cerebrospinal  fluids. 


General 

paralysis 

Tabes 

Cerebrospinal 
syphilis 

No. 
of  cases 

P.  ct. 

+ 

No. 

of  cases 

P.  ct. 

+ 

No. 
of  cases 

P.  ct. 

+ 

Marie  and  Levaditi 

39 
30 
45 
60 
41 

8 
54 

? 

35 
64 
56 

73 
93 
88.8 
73 
88 
100 
90 
90 

94 
92.1 

87.5 

9 

5 
11 

? 

12 
15 

66.6 

60 
54.5 

50 

66.6 

63 

8 
6 

4 
16 

Marie,  Levaditi,  and  Yamanouchi 
Stertz 

0 

Noguchi  and  Moore 

50 

Wassermann  and  Plant 

Morgenroth  and  Stertz 

Plant * 

0 

Nonne 

25 

Schlitze 

Marinesco 

Smith  and  Candler 

Noguchi,  Rosanoff,  and  Wiseman 

432 

90 

52 

56.2 

34 

19 

Table  14. — Examinations  of  blood-serum  and  cerebrospinal  fluid  in 
cases  of  leprosy. 


Serum 

Spinal  fluid 

No. 
of  cases 

Per  cent. 

+ 

No. 
of  cases 

Per  cent. 

+ 

Eitner 

2 
1 
26 
21 
26 
10 
10 
60 

100 

100 

100* 
57* 
30* 
50t 
70 
53 

19 
20 

Wechselmann  and  Meier 

Slatineanu  and  Danielopolu 

72 

Do 

0 

Jundell,  Almqvist,  and  Sandman 

Bruck  and  Gessner 

Noguchi 

Fox 

146 

61 

39 

86 

*  Including  weak  reactions.       t  Five  out  of  seven  cases  of  tubercular  form. 


SERUM  DIAGNOSIS  OF  SYPHILIS.  129 

Certain  investigators  have  reported  a  high  per- 
centage of  positive  reactions  in  leprosy  (as  will  be 
seen  by  perusing  Table  14)  and  in  other  nonspecific 
diseases,  notably  scarlet  fever  (Table  15),  carcinoma, 
and  diabetes  mellitus.^  In  Table  16  the  writer  shows 
a  study  of  the  results  of  322  cases  in  which  sj^philis 
does  not  play  an  etiological  part.  In  8  cases  of  pel- 
lagra Bass  reported  positive  reactions  with  the  Was- 
sermann  system,  but  later  investigations  by  Fox  on 
30  cases  and  by  Litterar  on  20  cases  of  the  same 
disease  examined  with  my  system  failed  to  confirm 
Bass's  results. 

In  Tables  17  and  18  the  results  of  examination 
of  132  cases  of  diseases  in  which  syphilis  may  be  an 

^  A  few  investigators  obtained  positive  reactions  in  an  astonishingly 
large  proportion  of  nonsyphilitic  cases,  while  the  majority  of  the  work- 
ers do  not  get  such  results.  Among  those  who  reported  a  large  number 
of  positive  reactions  in  nonsyphilitic  cases  may  be  mentioned  Weil 
and  Braun  and  Elias,  Neubauer,  Porges,  and  Salomon.  Weil  and 
Braun  encountered  4  positive  in  12  cases  of  pneumonia,  3  positive  out 
of  20  cases  of  typhoid  fever,  2  positive  out  of  21  cases  of  tuberculosis, 
1  positive  out  of  4  cases  of  diabetes  mellitus,  and  2  positive  in  11  cases 
of  tumors.  Elias  and  others  found  5  positive  in  33  cases  of  tuberculosis 
and  4  positive  in  14  cases  of  tumors.  Hancken  met  with  2  positive 
reactions  in  28  control  cases,  one  being  a  subject  with  scarlatina  and 
one  other  with  diphtheria.  Lohlein  examined  250  cases  and  obtained 
positive  results  in  4  cases  of  tuberculosis  and  carcinoma.  Later  inves- 
tigators, especially  those  who  had  been  working  with  the  reaction  con- 
stantly, all  failed  to  get  such  results  as  are  presented  above,  if  not 
absolutely  free  from  getting  an  occasional  weak  positive  reaction  in 
cases  of  carcinoma,  scarlet  fever,  or  diabetes.  It  should  be  suspected 
that  when  one  obtains  a  high  percentage  of  positive  reactions  in  non- 
syphilitic cases  he  is  not  doing  the  test  properly. 
10 


130 


SERUM  DIAGNOSIS  OF  SYPHILIS. 


Table  15. — Cases  of  scarlatina. 


No. 
of  cases 

P.  Ct. 

+ 

No. 

of  cases 

P.  ct. 

+ 

Much  and  Eicheberg. . 

130 
33 

10 

52 
37 

46 

0 

50* 

i.st 

2.5t 

Seligmann  and  Klop- 
stok  

30** 
61 
28 
63 
57 
106 

Jochmann  and  Topfer 
Halberstadter,  Muller, 

Boas  and  Hauge 

Bruck  and  Cohn 

Noguchi 

1.5t 

0 
1.5t 
25  § 

Meier 

Fua  and  Koch 

Hecht 

1 

*  Weak  reactions  only,  which  gave  negative  results  when  tested  with  several  other 
extracts. 

**  All  negative  in  13  cases  examined  on  July  1-3.  but  16  additional  cases  examined 
one  month  later  with  the  same  antigen  gave  3  weak  and  13  strong  positive  reactions. 
These  investigators  are  inclined  to  think  that  their  antigen  altered  on  standing,  hence 
the  positive  results. 

tOne  case  showed  some  inhibition.  The  case  of  Noguchi  was  subsequently  proven 
to  be  a  child  with  congenital  lues. 

§  Weak  reactions  only,  which  finally  disappeared  on  standing. 

Table  16. — Noguchi  system.    Cases  in  which  syphilis  can  be  excluded  with 
a  fair  degree  of  certainty. 


Cases 
examined 


Carcinoma 

Sarcoma 

Adenosarcoma 

Endothelioma 

Scarlatina 

Varicella 

Measles 

Tuberculosis 

Lupus 

Banti's  disease 

Hodgkin's  disease 

Muscular  distrophy. 

Neurasthenia 

Dementia  prsecox. . .  . 
Various  skin  diseases 
Miscellaneous 


51 
3 
1 
1 

62 
1 

2 

52 

2 

1 

2 

5 

2 

5 

58 

74 

322 


50 
3 
1 
0 

60 
1 
2 

52 
2 
0 
2 
5 
2 
5 

58 

74 

317 


SERUM  DIAGNOSIS  OF  SYPHILIS. 


131 


etiological  factor  and  of  130  cases  of  eye  diseases  of  all 
sorts  studied  by  Martin  Cohen  and  Bronfenbrenner 
are  given. 

In  certain  nervous  diseases  of  unknown  origin  the 
Wassermann  reaction  has  been  resorted  to  as  a  means 
of  determining,  if  possible,  the  nature  of  the  causa- 

Table  17. — Noguchi  system.    Cases  in  which  syphilis  is  an  etiological  factor 
or  cannot  be  excluded  as  a  possible  cause  of  the  condition. 


Cases 
examined 

+ 

- 

± 

Cirrhosis  of  liver 

7 

21 

1 

10 

29 

5 

82 

4 

8 

2 

8 

3 

2 

5 
11 

1 

2 
14 

1 

1* 

1 

4 

1 

3 

2 

0 

1 
9 
0 
6 

15 
4 

31 
3 
4 
1 
5 
0 
2 

1 

Ascitic  fluids 

0 

Aortic  insufficiency 

0 

Chronic  arthritis 

2 

Eye  cases 

0 

Diabetes 

0 

Eczema 

0 

Scleroderma 

0 

Brain  tumor  (?) 

0 

Central  gliosis  (?) 

0 

Hemiplegia 

0 

Spastic  parapleeia 

1 

Raynaud's  disease  f 

0 

132 

46 

81 

4 

*This  case  has  been  reported  also  by  Fox,  in  Table  8.  For  the  other  31  cases  I  am 
indebted  to  Dr.  Daisy  Orleman-Robinson. 

+  Kaiiski  and  Buerger,  using  Wassermann's  and  Noguchi's  systems,  got  negative 
results  in  16  cases  of  thrombo-angiitis  obliterans. 

tive  factor.  Thus,  Raviart,  Breton  and  Petit  ex- 
amined various  forms  of  insanity,  aside  from  para- 
syphilitic  patients,  in  regard  to  the  presence  of  this 
reaction  in  the  blood.  Their  results  are  somewhat 
striking,  as  they  got  positive  reactions  in  about  30 
to  40  per  cent,  of  cases  of  epilepsy,  idiocy,  and  im- 


132 


SERUM  DIAGNOSIS  OF  SYPHILIS. 


becility.     In  three  of  five  cases  of  dementia  senilis 
and  in  five  out  of  19  cases  of  dementia  prsecox  they 

Table  18. — Noguchi  system.     (Cohen  and  Bronfenhrenner .) 


1. 

2. 

3. 

4. 

5. 

6. 

7. 

8. 

9. 
10. 
11 

12. 
13. 
14. 
15. 
16. 
17. 
18. 
19. 
20. 
21. 


24. 


Interstitial  keratitis 

Iritis 

Irido-cyclitis 

Optic  neuritis 

Optic  atrophy 

Neuro-retinitis 

Retrobulbar  neuritis .... 
Retinitis  pigmentosa. .  .  . 

Retinitis 

Detachment  of  retina .  .  . 
Embolism  of  central  ar- 
tery  

Chorioiditis 

Chorio-retinitis 

Scleritis 

Ophthalmoplegia  interna 
Oculomotor  paralysis..  . 

Ptosis 

Paralysis  external  rectus 

Diplopia 

Corneal  ulcer 

Chancre  of  upper  lid ...  . 
Sympathetic  ophthalmia 

Acromegaly 

Amaurotic  family  idiocy. 
Graves'  disease 


Total 
Number  of 

Cases 


38 

16 

3 

10 

10 

6 

1 

8 

1 

2 

2 
8 
6 


130 


Undoubtedly 
Syphilitic 

+       — 


14     15* 


Doubtfully 
Syphilitic 

+      - 


17     12 
5       6 


46     55 


Under 

Recent 

Treatment 

+      — 


14     18 


♦  Under  antisyphilitic  treatment. 

got  positive  results.  Raubinovitch  and  Levaditi  ex- 
amined 15  cases  of  dementia  prascox  and  got  positive 
results  in  20  per  cent,  with  the  blood,  but  all  the 


SERUM  DIAGNOSIS  OF  SYPHILIS. 


133 


spinal  fluids  examined  gave  negative  results.  This 
last  fact  agrees  with  the  observation  of  the  writer  and 
Moore.  Rosanofl^,  Wiseman,  and  the  writer  exam- 
ined 413  cases  of  various  forms  of  insanity  for  the 

Table  19. — Psychiafric  cases. 


Clinical  diagnosis 


Arteriosclerotic  dementia  . 

Brain  tumor 

Traumatic  psychosis 

Senile  dementia 

Infant,  cerebr.  palsy 

Epilepsy 

Huntington's  chorea 

Ursemic  psychosis 

Alcoholic  psychosis 

Polyneuritic  psychosis  .... 
Involution  melancholia  . , . 

Dementia  prsecox 

Manic  depressive  insanity . 

Paranoiac  condition 

Imbecility 

Constitutional  inferiority.  . 
Unclassified  


1 

Blood- 

seruir 

I        1 

Cerebrospinal  fluid 

O    g 

a  2 

Reactions 

1? 

Reactions 

- 

+ 

± 

- 

+ 

± 

10 

10 

0 

0 

9 

9 

0 

0 

1 

1 

0 

0 

1 

1 

0 

0 

1 

1 

0 

0 

1 

1 

0 

0 

16 

13 

1 

2 

10 

8 

1 

1 

6 

6 

0 

0 

5 

5 

0 

0 

69 

48 

12 

9 

55 

50 

3 

2 

2 

1 

1 

0 

1 

1 

0 

0 

1 

1 

0 

0 

1 

1 

0 

0 

9 

4 

2 

3 

6 

4 

1 

1 

8 

7 

1 

0 

8 

8 

0 

0 

10 

8 

2 

0 

7 

7 

0 

0 

131 

99 

15 

17 

83 

76 

3 

4 

14 

9 

2 

3 

7 

5 

2 

0 

9 

7 

1 

1 

4 

4 

0 

0 

6 

4 

2 

0 

6 

6 

0 

0 

1 

1 

0 

0 

1 

1 

0 

0 

40 

28 

6 

6 

38 
243 

35 

222 

2 
12* 

1 
9* 

334 

248 

45* 

41* 

■piS 


15 


*  These  cases  showing  positive  and  doubtful  reactions  may  have  had  syphilis,  but 
it  was  difBcult  to  ascertain  the  disease  in  all  the  cases.  In  15  cases  at  least,  syphilis 
was  proven  to  be  present. 

reaction  in  serum  and  cerebrospinal  fluid  and  obtained 
the  results  similar  to  those  of  previous  investigators. 
Atwood  and  Bronfenbrenner,  using  the  Noguchi 
system,  examined  204  cases  of  idiots  and  found  14.7 
per  cent,  positive  reactions  among  them. 


134  SERUM  DIAGNOSIS  FOR  SYPHILIS. 

A  rough  classification  of  the  cases  is  shown  in 
the  following  list: 

CLASSIFICATION  OF  THE  204  LOW-GRADE  IDIOTS  TESTED 

Idiopathic  idiots 120 

Diplegias 47 

Hemiplegics 7 

Epileptics  without  paralysis 13 

Hydrocephalics 5 

Microcephalics 6 

Cretins 2 

MyxcEdematous 1 

Amaurotic  family  idiocy 1 

Idiocy  with  cerebellar  ataxia 2 

Several  (4)  of  the  patients  were  blind,  and  several 
(4)  mute.  There  were  other  physical  disorders,  not 
syphilitic,  in  other  cases. 

CLASSIFICATION  OF  THE  30  IDIOTS  WHO  SHOWED  A  POSITIVE 
SEROREACTION 

Positive.      Total.      Percentage. 

Idiopathic 13  120  10 

Diplegics 11  47  23 

Hemiplegics 2  7  28 

Microcephalics 1  5  20 

Epileptic  without  paralysis 1  12  8 

Cerebellar  ataxics 2  2  100 

Still  further  analyzed,  one  of  the  diplegics  with 
positive  reaction  was  epileptic,  one  hydrocephalic,  and 
one  epileptic  with  mutism.  One  of  the  cerebellar 
ataxic  patients  was  microcephalic,  and  one  of  the 
diplegics  was  blind.  One  out  of  four  deaf-mutes 
showed  a  positive  seroreaction.  The  myxedematous 
idiot,  the  two  cretins,  and  the  patient  with  amaurotic 
family  idiocy  showed  negative  reactions. 

The  percentage  of  positive  reactions  found  was 
much  greater,  in  proportion,  in  idiots  with  superadded 


SERUM  DIAGNOSIS  OF  SYPHILIS.  135 

gross  organic  brain  defect  than  that  found  in  idio- 
pathic idiocy. 

Their  results  are  in  close  agreement  with  those  of 
Lippmann,  who  employed  the  Wassermann  system 
in  Germany. 

In  gynaecological  conditions  the  reaction  has  also 
been  called  upon  to  test  the  validity  of  the  laws  of 
Colles  and  Prof  eta.  Miiller  found  that  with  the  blood 
of  wives  of  syphilitic  husbands,  where  the  former  had 
repeated  abortions  and  premature  births,  the  results 
were  usually  negative  and  that  their  oifspring  also 
gave  negative  reaction.  Knopf elmacher  and  Lehn- 
dorffer  examined  32  apparently  healthy  mothers  of 
syphilitic  children  and  obtained  positive  reactions  in 
18.  Halberstadter,  Miiller  and  Reiche  found  that  the 
reaction  may  be  negative  with  children  of  syphilitic 
mothers,  and  vice  versa,  while  Boas  and  Thomsen 
assert  that  the  reaction  can  develop  later  in  children 
whose  blood  gives  a  negative  result  at  the  time  of 
birth.  They  all  agree  that  the  negative  reaction  in 
these  children  or  mothers  is  largely  due  to  the  latency 
of  the  disease,  but  is  not  a  sign  of  immunity  against 
the  disease.  Thus,  while  the  mother  of  a  syphilitic 
infant  may  present  no  sign  of  syphilis,  yet  examina- 
tion of  the  blood  of  the  mother  gives  positive  reaction 
in  half  the  number  of  cases  examined.  However, 
much  more  has  to  be  done  before  the  dictum  of  Colles 
can  be  overthrown. 


XII. 

EFFECT  OF  TREATMENT  UPON  THE  REACTION. 

MERCURIAL    TREATMENT. 

Much  work  has  been  done  by  numerous  investiga- 
tors to  determine  the  result  of  various  forms  of  treat- 
ment upon  the  syphilitic  antibodies  in  the  blood,  and 
it  would  seem  that  the  time  has  not  yet  come  to  make 
a  dogmatic  statement  upon  this  subject.  It  is  known, 
however,  that  the  reaction  frequently  disappears  after 
a  short  course  of  treatment,  as  will  be  pointed  out  be- 
low, often  to  return  again  within  a  greater  or  lesser 
period  of  time. 

Citron,  who  was  among  the  first  to  investigate 
the  effect  of  treatment  upon  the  reaction,  found  that 
whereas  before  treatment  the  percentage  of  positive 
results  obtained  by  him  was  81,  treatment  had  the 
effect  of  reducing  the  figures  to  65  per  cent.  In 
about  half  of  these  cases,  numbering  57  in  all,  but 
one  course  of  treatment  was  given.  Bruck  and  Stern 
obtained  positive  reactions  in  81.5  per  cent,  of  173 
untreated  cases,  and  in  another  group  of  treated  cases 
got  positive  reactions  in  only  28  per  cent.  Blaschko 
studied  52  positive  cases  of  manifest  syphilis  and 
after  treatment  45  of  these  gave  negative  results; 
of  38  cases  of  latent  syphilis  31  gave  negative  results 
after  treatment.  Hoehne  studied  211  cases  which  be- 
fore treatment  gave  positive  reactions,  and  found  that 

136 


SERUM  DIAGNOSIS  OF  SYPHILIS.  137 

in  5Q  per  cent,  the  reaction  disappeared  after  thera- 
peutic interference,  but  in  spite  of  some  treatment 
33.9  per  cent,  gave  a  positive  reaction.  In  five  cases, 
after  eleven  to  twelve  injections  of  mercuric  salicylate 
over  a  period  of  two  months,  the  reaction  was  positive. 
Lesser  states  that  a  positive  reaction  can  be  made 
negative  in  about  35  per  cent,  of  cases  by  giving  30 
inunctions  of  mercury,  12  injections  of  an  insoluble 
mercuric  preparation,  or  25  injections  of  a  soluble 
mercuric  preparation.  The  rapidity  with  which  the 
reaction  disappears  is  very  variable  in  diiFerent  in- 
dividuals. Boas  found  that  after  a  course  of  injec- 
tions over  two  or  three  months  the  reaction  became 
negative  in  76  out  of  82  cases,  and  states  that  the 
reaction  may  return  within  a  month  after  cessation 
of  treatment,  indicating  a  recurrence. 

V.  C.  Pedersen  followed  carefully  the  course  of 
treatment  on  a  large  number  of  cases  by  means  of 
my  system.  The  clinical  classification  of  cases  bj'' 
this  author  is  most  elaborate  and  points  out  very 
clearly  how  the  reaction  stands  in  relation  to  the 
clinical  side  of  these  cases. 

In  order  to  fulfil  the  hope  of  tracing  the  progress 
of  this  test,  his  records  have  been  divided  into  the 
following  classes.  It  will  be  seen  at  once  that  this 
refinement  of  study  results  in  the  apparent  circum- 
stance of  having  a  small  number  of  observations  in 
each    class.      When,    however,    the    class    titles    are 


138  SERUM  DIAGNOSIS  OF  SYPHILIS. 

studied,  it  will  be  seen  that  without  these  or  similar 
designations,  it  would  be  almost  impossible  to  study 
the  work  more  or  less  minutely. 
The  classes  are  as  follows: 

Class  A.  Chancre  unhealed;  no  secondary  symptoms. 

Group  I.  No  local  or  systemic  treatment. 

Group  II.  Local  or  systemic  treatment. 
Class  B.  Chancre   unhealed;   secondaries   out. 

Group  I.  No  local  or  systemic  treatment. 

Group  II.  Local  or  systemic  treatm-ent. 
Class  C.  Chancre  healed  recently;  no  secondaries  out. 

Group  I.  No  local  or  systemic  treatment. 

Group  II.  Local  or  systemic  treatment. 
Class  D.  Chancre   healed;    secondaries   out;    duration   of   infection   less 
than  six  months. 

Group  I.  No  local  or  systemic  treatment. 

Group  II.  Local  or  systemic  treatment. 
Class  E.  Duration  of  infection   six  to  twenty- four  months;   symptoms 

absent.     Treatment  given. 
Class  F.  Duration  of  infection   six  to  twenty- four  months;   symptoms 

present.     Treatment  absent,  imperfect  or  irregular. 
Class  G.  Tertiary  period;  infection  older  than  twenty- four  months. 

Group  I.  No  symptoms  present. 

Group  II.  Lesions  of  mucosa,  skin  and  appendages  of  the  skin. 

Group  III.  Lesions  of  cartilage,  bone,  joint,  and  muscle. 

Group  IV.  Lesions  of  glands  and  viscera. 

Group  V.  Lesions  of  the  nervous  system. 
Class  H.  Diagnosis  of  syphilis  positive.    Records  otherwise  too  defective 
for  accurate  classification  under  any  of  the  foregoing  subdivisions. 
Class  I.  Comparison  of  all  tests  repeated  two  or  more  times. 
Class  J.  Uncertain  clinical  diagnosis  of  syphilis. 

Taylor  gives  Ricord's  subdivision  of  syphilis  into 
three  periods — primary,  secondary,  and  tertiary — 
which  is  classic  and  accepted.  The  primary  stage  is 
usually  separated  into  two  periods  of  incubation, 
the  first  period  of  incubation  being  the  time  elapsing 
between    infection    and    the    development    of    hard 


SERUM  DIAGNOSIS  OF  SYPHILIS. 


139 


chancre,  and  the  second  incubatory  period  including 
the  time  between  the  development  of  the  chancre  and 
the  appearance  of  the  secondary  lesions.  The  second 
stage  of  syphilis  usually  embraces  the  first  two  years, 
and  the  tertiary  period  begins  at  the  end  of  the  second 
year. 

The  results  obtained  by  Pedersen  are  given  below : 

Table  20. —  The  relation  in  -percentages  between  the  various  degrees  of 
the  syphilis  reaction  {Noguchi  system)  from  class  to  class. 


Classes 

and 
groups. 

Total 
number 
of  cases. 

Num- 
ber. 

Num- 
ber. 

± 

Num- 
ber. 
<<  + 

Num- 
ber. 

<  + 

Niim- 
ber. 

+ 

Num- 
ber. 

+  4- 

Percentage 

positive  test 

(approximate). 

A  I.... 

16 

1 

1 

4 

10 

94 

A  11... 

3 

2 

1 

100 

B  I.... 

11 

1 

5 

5 

100 

B  II... 

5 

1 

1 

3 

100 

G  I.... 

C  II... 

1 

1 

100 

D  I.... 

12 

1 

4 

7 

100 

D  II... 

23 

3 

1 

7 

6 

6 

83 

E 

31 

7 

2 

2 

12 

6 

2 

71 

F  

13 

2 

3 

3 

5 

85 

G  I.... 

25 

12 

1 

2 

6 

4 

48 

G  II... 

18 

3 

3 

3 

6 

3 

67 

G  III.. 

10 

3 

2 

3 

2 

50 

G  IV... 

7 

3 

1 

1 

1 

1 

43 

G  v.... 

12 

3 

3 

6 

75 

It  will  be  noticed  that  Pedersen's  classification 
for  his  series  of  observations  closely  follows  the 
original  designation  of  Ricord.  In  order,  however, 
to  permit  study  of  the  effects  of  treatment,  it  was 
deemed  wise  to  allow  six  months  to  elapse  before 
Class  D  was  enumerated  to  embrace  chiefly  cases 
of  later  secondary  syphilis  along  with  Classes  E 
andF. 


140 


SERUM  DIAGNOSIS  OF  SYPHILIS. 


As  the  records  of  Classes  H  and  J  lacked  in 
necessary  details  for  being  embodied  in  the  above 
table  they  will  not  be  quoted  here,  while  a  brief  sum- 
mary of  results  is  made  by  Pedersen  with  Class  I. 

Class  I  is  a  comparison  of  all  tests  repeated  upon 
a  given  patient  two  or  more  times,  from  which  it  is 
hoped  to  point  out  better  than  the  foregoing  classes 
do  the  direct  tendency  of  reaction  to  disappear  under 
treatment  and  with  the  lapse  of  time.  In  this  class 
are  sixty-two  observations.  In  the  majority  of  these 
observations  it  is  found  that  the  second  tests  always 
show  a  decrease  in  the  reaction,  provided  treatment 
was  active  and  efficient.  Occasionally,  the  total  dis- 
appearance of  the  test  is  recorded. 

Craig  (U.  S.  A.  Med.  School)  has  gathered  the 
following  data: 

Table  21. — The  result  of  specific  treatment  upon  the  syphilis  reaction 
(Noguchi  system). 


Time  treated 

No.  of  cases 

Positive 

Negative 

Time  treated 

No.  of  cases 

Positive 

Negative 

2  weeks 

2 

1 

1 

40  weeks 

1 

1 

0 

4     • 

1 

0 

1 

1  year 

4 

1 

3 

6     ' 

2 

1 

1 

14  months 

3 

1 

2 

8     ' 

3 

2 

1 

15       •• 

1 

1 

0 

10     ' 

1 

1 

0 

18      '• 

2 

2 

0 

12     ' 

1 

3 

1 

2  years 

6 

5 

1 

16     ' 

4 

1 

0 

1  30  months 

1 

1 

0 

24     • 

3 

3 

1 

3  years 

2 

0 

2 

28     • 

4 

1 

2 

8     " 

1 

1 

0 

82     ' 

5 

1 

3 

12     " 

1 

1 

0 

36     • 

4 

1 

Total  cases  with  treatment,  52.    Positive,  32.    Negative,  20. 


SERUM  DIAGNOSIS  OF  SYPHILIS.  141 

Craig  arrived  at  the  conclusion  that  the  disap- 
pearance or  reduction  of  the  reaction  during  the  treat- 
ment is  a  valuable  indicator  for  the  effectiveness  of 
the  treatment.  Irregular  and  inadequate  therapeutic 
measure,  no  matter  how  long  a  period  it  may  be 
extended,  leaves  the  reaction  still  positive. 

From  the  above  it  will  be  seen  that  the  reaction  is 
affected  greatly  by  the  treatment  of  the  disease,  but 
that  some  cases  frequently  persist  in  giving  a  positive 
reaction  in  spite  of  what  is  done  for  them.  In  heredi- 
tary lues  the  reaction  is  difficult  to  get  rid  of,  often 
persisting  in  spite  of  most  rigorous  interference.  The 
reaction  may  return  shortly  after  cessation  of  treat- 
ment, so  that  it  may  be  necessary  to  make  frequent 
tests  to  determine  whether  further  therapeusis  is  in- 
dicated. While  it  seems  settled  among  the  profession 
that  a  positive  reaction  in  a  syphilitic  case  is  an  in- 
dication for  additional  treatment,  it  is  not  definitely 
established  that  the  disappearance  of  the  reaction  is 
justification  for  the  cessation  of  treatment,  especially 
as  the  reaction  may  be  quickly  affected  by  treatment. 

SALVARSAN    (OR   606)    TREATMENT. 

The  influence  of  the  treatment  of  syphilis  with 
Ehrlich-Hata  dioxydiamidoarsenobenzol  or  so-called 
"606"  upon  the  serum  reaction  has  been  studied  by 
various  investigators. 


142  SERUM  DIAGNOSIS  OF  SYPHILIS. 

Nichols  treated  the  rabbits  and  monkeys  experi- 
mentally inoculated  with  syphilis  and  yaws  by  means 
of  intravenous  injection  of  the  arsenobenzol  and  found 
that  the  spirochaetae  disappear  within  24  hours  and 
the  serum  reaction  becomes  negative  within  about 
four  weeks  after  the  injection. 

The  observations  upon  human  subjects  have  been 
made  by  a  large  number  of  investigators  on  a  vast 
syphilitic  material  in  different  clinics  and  hospitals. 
Although  still  too  early  to  judge  its  efficacy  in  the 
treatment  of  syphilis  this  preparation  has  already 
done  much  in  clearing  up  the  syphilitic  lesions  within 
a  comparatively  short  period.  It  is  remarkable,  in- 
deed, that  relapses  of  the  disease  after  disappearance 
of  symptoms  by  a  single  injection  have  been  com- 
paratively few. 

I  will  review  very  briefly  the  literature  on  "606" 
with  special  reference  to  the  number  of  relapses  and 
the  effect  of  the  treatment  upon  the  Wassermann 
reaction. 

Wechselmann  treated  1250  cases  and  observed 
only  40  relapses.  The  serological  study  of  Wechsel- 
mann's  cases  was  conducted  by  Lange,  who  analyzed 
268  cases  in  detail.  They  found  that  153  out  of  268 
cases  became  negative  in  five  weeks  after  the  injec- 
tion. There  were  18  cases  which  were  negative 
before  and  after  the  injection.     The  reaction  was 


SERUM  DIAGNOSIS  OF  SYPHILIS.  143 

still  positive  in  97  cases  when  examined  at  the  end 
of  five  weeks,  although  a  more  or  less  reduction  in 
the  strength  of  the  reaction  was  noticed  in  34  of 
these  cases. 

Michaelis  treated  110  cases  and  observed  3  re- 
lapses. According  to  him  the  serum  reaction  may 
become  negative  within  a  period  of  from  two  to  ten 
weeks  after  the  injection.  In  a  few  instances  the 
reactions  became  stronger  than  before  the  injection 
while  the  clinical  symptoms  were  fast  disappearing 
under  the  influence  of  "606."  This  last  phenomenon 
has  also  been  observed  by  Munk  and  Fraenkel  and 
Grouven. 

Herxheimer  treated  789  cases  and  had  33  relapses 
within  a  period  of  five  months  of  observations.  Many 
of  his  cases  went  out  of  sight  before  a  negative  reac- 
tion was  obtained.  In  a  limited  number  of  cases 
which  remained  under  his  observation  for  a  sufficient 
length  of  time  the  reaction  became  negative  in  75 
per  cent,  of  cases  within  50  days  after  the  injection. 
Bering  reports  that  the  reaction  became  negative  in 
40  cases  after  the  lapse  of  five  weeks  or  longer,  while 
it  remained  positive  in  26  cases. 

Treupel,  Halberstadter,  Ledermann,  Schlesinger, 
Bruhns,  and  Hoffmann  experienced  very  slow  reduc- 
tion or  persistence  of  the  reaction  in  most  of  their 
patients.    On  the  other  hand,  Kromayer,  Gennerich, 


144  SERUM  DIAGNOSIS  OF  SYPHILIS. 

Linser,  Cramer,  and  others  saw  the  reaction  disap- 
pear in,  at  least,  50  per  cent,  of  the  cases  within 
four  to  five  weeks. 

In  the  work  above  quoted  the  arsenobenzol  was 
used  either  in  a  neutral  or  a  slightly  alkaline  sus- 
pension and  administered  intramuscularly  by  one  set 
and  subcutaneously  by  the  other  set  of  investigators. 
In  the  beginning  most  of  the  earlier  cases  had  re- 
ceived only  0.3  gram  as  a  dose  (instead  of  0.6  gram 
as  recommended  later). 

An  intravenous  administration  of  "606"  had  been 
advocated  by  Schreiber,  who  compared  the  efficacy 
of  the  arsenobenzol  by  giving  it  intramuscularly  in 
one  series  and  intravenously  in  another  series  of 
cases.  Schreiber  administered  "606"  intramuscu- 
larly to  152  cases  and  observed  18  relapses,  while  in 
5Q5  cases  treated  by  the  intravenous  injection  there 
was  only  one  case  of  recidive.  In  his  earlier  report 
Schreiber  mentions  that  the  reaction  disappeared 
in  50  per  cent,  of  cases  after  the  injection  (intra- 
muscular as  well  as  intravenous?) ,  but  inferring  from 
his  last  report  he  now  seems  to  succeed  in  making  the 
reaction  disappear  in  every  case  by  the  intravenous 
injection,  because  he  orders  another  intravenous 
injection  whenever  the  reaction  remains  positive  after 
the  first  administration.  Geronne  treated  220  cases 
with     intravenous     administration     of    "606"    and 


SERUM  DIAGNOSIS  OF  SYPHILIS.  145 

observed  14  recidives.  Of  77  cases  of  all  stages  of 
syphilis  the  reaction  became  negative  in  37  and 
remained  so  for  at  least  8  weeks,  while  in  the  remain- 
ing 40  it  became  negative  for  a  time  and  later  again 
positive.  In  9  out  of  13  recidives  the  reaction  quickly- 
disappeared  upon  a  second  injection,  while  the  rest 
retained  the  positive  reaction  after  the  second  dose. 
In  going  over  the  entire  literature  on  the  Ehrlich- 
Hata  "606"  I  find  that  the  statements  in  regard  to 
the  Wassermann  reaction  are  extremely  brief  and 
vague,  and  it  was  with  much  difficulty  that  I  could 
gather  the  data  just  reviewed.  The  chief  reason  for 
the  incomplete  serological  analysis  of  their  cases  is 
unquestionably  due  to  the  enormous  labor  and 
material  demanded  by  the  original  Wassermann 
method.  It  is  by  no  means  easy  to  carry  out  a  syste- 
matic quantitative  serological  examination  on  a  very 
large  number  of  cases  by  this  method.  Even  when 
this  was  done  the  results  would  not  permit  one  to 
gain  an  insight  into  the  quantitative  relation  of  the 
reaction  to  the  clinical  course  of  the  disease  under 
the  treatment  with  "606,"  because  the  results  obtained 
with  the  original  method  do  not  indicate  differences 
among  positive  reactions  as  to  whether  the  complete 
fixation  is  caused  by  exactly  one  antibody  unit  or  by 
several  or  any  other  number  above  one. 

In  order  to  follow  the  effect  of  "606"  upon  the 
11 


146  SERUM  DIAGNOSIS  OF  SYPHILIS. 

serum  reaction  in  a  strictly  quantitative  manner  I 
have  undertaken,  with  Dr.  Bronfenbrenner,  a  series  of 
examinations  of  cases  treated  with  the  arsenobenzol. 
The  cases  ^  included  in  my  series  belonged  to  Drs. 
Cohen,  Fordyce,  Fox,  Fuller,  Gottheil,  Henderson, 
Kakels,  Lapowsky,  Luckett,  Pedersen,  Pollitzer,  and 
Simonds.  The  cases  of  Dr.  Fuller  and  Dr.  Gottheil 
were  injected  by  me. 

The  total  number  of  cases  available  for  a  complete 
serological  analysis  is  102.  More  than  half  of  this 
number  have  been  under  observation  for  a  period  over 
three  months,  while  the  latest  cases  were  injected 
about  four  weeks  ago.  The  amounts  of  the  arseno- 
benzol were  0.5  to  0.6  for  men,  0.4-0.5  for  women 
and  0.015-0.025  for  children.  The  modes  of  injec- 
tion were  variable  with  different  investigators,  but 
the  majority  injected  a  slightly  alkaline  suspension 
or  semi-solution  intramuscularly  or  subcutaneously. 
Occasionally  intravenous  injections  were  made,  espe- 
cially in  later  cases.  Many  cases  have  been  injected 
into  the  lumbar  sacral  region  of  the  erector  spinse 
muscle  as  recommended  by  Meltzer.  From  my 
experience  I  consider  the  intravenous  injection  of  an 
alkaline  solution  as  the  best,  and  the  subcutaneous 
administration  the  worst.     The  intramuscular  mode 

1  For  their  courtesy  in  permitting  me  to  use  their  cases  for  the  present 
study  I  express  my  thanks  to  these  gentlemen. 


SERUM  DIAGNOSIS  OF  SYPHILIS.  147 

of  injection  into  the  glutei,  though  decidedly  less 
effective  than  the  intravenous  one,  gives  a  good 
result.  Whether  Meltzer's  method  of  intramuscular 
injection  is  equally  effective  as  the  intravenous  can- 
not be  told  yet,  but  if  this  proves  to  be  the  case  his 
method  may  be  adopted. 

The  quantitative  determination  of  the  serum  reac- 
tion in  each  case  was  carried  out  with  my  method  in 
the  manner  already  described  under  the  titration  of 
syphilitic  antibody.  (See  page  69.)  The  blood 
was  examined  before  the  injection  and  then  1  day, 
3  days,  1  week,  2  weeks,  3  weeks,  4  weeks,  6  weeks, 
8  weeks,  etc.,  after  the  injection. 

Table  22  shows  the  serological  conditions  of  these 
cases  before  and  some  time  after  the  injection  of  the 
arsenobenzol.  This  table  furnishes  us  with  many  inter- 
esting facts  in  regard  to  the  syphilis  reaction. 

Examining  first  the  varieties  of  the  reactions  in 
these  102  strongly  positive  specimens  one  will  notice 
that  in  primary,  secondary,  tertiary  and  hereditary 
syphilis  the  average  specimens  contained  more  than 
one  antibody  unit,  more  frequently  2,  3,  and  4  units. 
Among  the  specimens  derived  from  secondary  syph- 
ilis one  with  10  units  was  encountered.  On  the 
other  hand,  the  majority  of  specimens  from  latent 
syphilis  contained  1  or  2  units.  In  cerebrospinal 
syphilis  and  tabes  the  antibody  content  was  also  com- 


148  SERUM  DIAGNOSIS  OF  SYPHILIS. 

paratively  low.  Taking  the  average  for  different 
stages  of  syphilis  in  groups  one  finds  that  of  the 
secondary  to  be  the  highest,  followed  by  those  of  the 
hereditary,  tertiary,  primary,  and  latent  syphilis. 
It  may  be  recalled  here  that  any  specimen  containing 
more  than  one  antibody  unit  is  capable  of  giving  a 
complete  inhibition  of  haemolysis,  commonly  known  as 
a  strong  positive  reaction. 

Let  us  next  study  the  serological  conditions  which 
have  been  created  in  the  same  series  of  cases  after  the 
injection  of  "606."  There  we  find  a  striking  contrast 
to  the  conditions  which  existed  before  injection.  There 
are  two  new  columns,  one  for  entering  the  number 
of  specimens  as  negative  and  the  other  for  the  speci- 
mens which  no  longer  contain  one  unit  of  antibody, 
and  both  are  well  filled  up  with  different  figures. 
In  the  following  columns  one  notices  that  beyond  the 
column  for  4  units  all  are  blank  up  to  the  last,  signify- 
ing, of  course,  that  after  the  treatment  none  of  the 
cases  contained  more  than  4  units.  In  reality  the 
positive  specimens  after  "  606  "  usually  contained  1 
or  2  units  and  seldom  3  or  4.  Speaking  more  in  detail, 
30  cases  lost  the  reaction,  24  cases  reduced  to  less  than 
one  antibody  unit,  while  the  remaining  48  (47-5  per 
cent.)  still  contained  more  than  one  unit  and  gave 
strong  positive  reactions.  When  speaking  vaguely 
these  48  cases  may  be  taken  as  an  evidence  that  the 


SERUM  DIAGNOSIS  OF  SYPHILIS. 


149 


H 

O 

w 

f* 

1^ 

OQ 

hj 

II 

H 

so 

a* 

:  3 

m 

! 

CO 

3 

fD 

§ 

p 

so 

3. 
3 

1 

■  f  • 

1 

1 

•3 

1 

i 

1 

1 

? 

1 

^ 

b: 

o 

CO 

CO 

h- 

Number  of  cases. 

to 

to 

C3> 

^ 

OJ 

00 

to 

These    102    c 
designated  as  sti 
tions,  but  note  th 
reaction    can    be 
number  of  the  a 
1  to  10  in  this  seri 

05 

to 

to 
to 
to 

CO 

CO 

to 

o> 
o 

00 
CO 

to 

to 
C;i 

to 

CO 

to 

to 

CO 

2? 

a- 
a 
1 
o 

s. 

a 

;ases 
rang 
at  a 
cau 
mtib 
es. 

o 

^ 

en 

CO 

*^ 

en 

OK 

tojj-. 

re' 

are 
ly  po 
stron 
ised   1 
lody 

to 

CO 

bO 

cr 

§ 
o 

g-^^^- 

_ 

o 

"t 

o 

00 
«5 

i 

o> 
o 

3  p  ct-3  2 

)_» 

CO 

L  '"' 

'-' 

o 

p' 

to 

to 

to 

CO 

to 

CO 

CO 

to 

o> 

o 

o 

o 

o 

^ 

tn 

ro 

Average  units. 

03 

~ 

30 

o 

00 

o 

05 

II  ?g-^ 

CO 

!_, 

!_, 

1 

^ 

These  48 
rongly  pos 
lie  they  ca: 
tive  methc 
47.5%.: 

1  Negativ 

2  Weakly 

1^ 

to 

o_ 

to 

-' 

lO 

to 

to 

in 

oo 

4^ 
00 

C7I 

CO 

1 

A 

c 
B 

n> 
•-1 
o 

> 

1  ca 
itiv 
n  b( 
id. 

e  re 
pos 

00 

j3 

w 

_. 

K 

*■ 

_ 

to 

■0 

s? 

E+ 

i-.-iB           <"  m  CO 

tJ' 

es  are  common 
reaction,  but  s 
when  analyzed 

,ction.    33.7%. 
tive  reactions. 

M 

^ 

to 

CO 

CD 

b; 

CO 

o 
o 

CO 

I*' 

05 

EJ-. 

cr 
o 
e. 
<< 

&■ 
B- 

"S' 

a 

o 

p 

2, 

ly  kn 
see  ho 
by  a 

19.8% 

o 
o 

00 

£!  ^  O 

o 

P  ^ 

to  <i  9 

o 

to 

b 
to 

DP 

o 

o 

o        c 

s      ► 

^ 

_. 

5 

D 

o 

3 

bo        c 

CO           c 

s  i 

J3 

s  ; 

1 

X 

Average  units. 

«l 

1 

1 

Compared  with  the 

^'^  " 

h    ^ 

w     rf^ 

r  ^ 

HJ   • 

[.. 

■"* 

original     (before 
injection). 

CO          c 
CO           c 

3        < 

Percentage  of  nega- 

3 

.o 

>3 

0 

^ 

tive    cases   after 

^ 

the 

injectio 

n.        II 

150  SERUM  DIAGNOSIS  OF  SYPHILIS. 

arsenobenzol  had  no  influence  upon  them,  but  the 
affair  changes  entirely  when  we  examine  the  data 
from  the  quantitative  side.  These  cases  while  still 
retaining  the  strong  reactions  are  decidedly  reduced 
in  antibody  content  from  what  they  originally  pos- 
sessed, as  will  be  seen  in  the  table.  For  the  sake  of 
comparison  I  have  calculated  out  the  average  anti- 
body units  for  each  group  of  cases  and  also  the  ratios 
between  the  averages  for  the  original  and  those  after 
the  treatment.  The  average  for  primary  cases  is  seen 
to  be  reduced  to  1/5,  that  for  secondary  to  1/3.5,  that 
for  tertiary  to  1/3.6,  that  for  latent  to  1/4,  that  for 
hereditary  to  1/2.7,  that  for  cerebrospinal  to  1/1.5 
and  that  for  tabes  to  1/4  of  the  original  antibody 
contents.  This  shows  clearly  that  the  arsenobenzol 
had  the  effect  of  reducing  the  antibody  content  in 
general. 

Now  considering  the  frequency  with  which  posi- 
tive reactions  became  negative  after  the  treatment  we 
find  that  in  40  per  cent,  of  the  primary  cases,  in  37 
per  cent,  of  secondary,  in  35  per  cent,  of  tertiary,  in 
33  per  cent,  of  latent,  in  14  per  cent,  of  hereditary  and 
in  50  per  cent,  of  incipient  tabes  the  reaction  was  lost, 
while  all  cases  of  cerebrospinal  syphilis  became  some- 
what weaker  but  remained  still  positive.  The  average 
of  the  negative  reactions  corresponds  to  33.7  per  cent, 
of  the  total  102  cases.- 


SERUM  DIAGNOSIS  OF  SYPHILIS. 


151 


The  percentage  of  weak  positive  eases  is  19.8  of 
the  total,  but  this  group  of  cases  is  progressively  los- 
ing the  strength  of  the  reaction  and  is  likely  to  fall 
in  due  course  of  time  into  the  category  of  the  nega- 
tive group. 

The  delation  Between  the  Clinical  and  Serological 
Findings. — In  order  to  study  what  relation  exists  be- 

Table  23. — Latent  cases  not  included. 


Symptoms 
cleared  up 

Slow 
improvement 

No 
improvement 

Relapses 

Primary  syphilis 

Secondary  syphilis 

Tertiary  syphilis 

Hereditary  syphilis 

Cerebrospinal  syphilis . . 
Tabes 

121 
5* 

7 
10 

2 

2 
3 

6 
1 

10 

1 

665 

20' 

12 

11 

1 5  became  negative.    ^  14  became  negative.    '  11  became  negative.    *  1  became  nega- 
tive.   ^31  became  negative.    ^  Became  negative.    '  1  became  negative. 

tween  the  clinical  data  and  the  serological  findings  a 
brief  summary  of  the  clinical  observations  is  presented 
above.  For  the  details  of  these  cases  I  refer  to  the 
publications  of  Fordyce,  Fox,  Gottheil,  Henderson, 
Kakels,  Luckett,  Pedersen,  and  PoUitzer. 

In  QQ  cases  which  responded  favorably  to  the 
arsenobenzol  and  lost  the  clinical  symptoms  within  a 
few  weeks  the  serum  reaction  became  negative  or 
reduced  in  strength.     The  effect  of  "606"  upon  the 


152 


SERUM  DIAGNOSIS  OF  SYPHILIS. 


clinical  symptoms  is  far  more  prompt  than  upon  the 
reaction.  In  a  large  number  of  cases  symptoms  com- 
mence to  clear  up  within  a  week,  not  infrequently, 
however,  even  within  24  hours  after  the  injection. 
The  progress  of  healing  is  usually  quick,  clearing  up 
within  a  few  weeks.  On  the  other  hand,  the  reaction 
becomes  gradually  weaker,  requiring  a  considerably 


Table  24. — Showing  the  length  of 
to  become  nega 

time  required  f 
tive  after  "606. 

or  positive 
>> 

reactions 

1 

95 

« 

to 

05 

00 

1 

1 

2 

1 

3 
3 

2 

1 

2 
5 
4 

3 

2 

1 

2 
1 

a 

Secondary  syphilis 

Tertiary  syphilis 

Latent  syphilis 

Hereditary  syphilis 

Cerebrospinal  syphilis  . . . 
Tabes 

1 

14 
11 
2 
1 
0 
1 

3 

10 

11 

5 

4 

1 

34 

longer  time  before  turning  into  a  negative.  In 
promptly  cured  cases  the  reaction  may  disappear 
within  two  weeks,  although  in  some  instances  it  may 
take  four  or  five  weeks.  In  this  group  only  31  cases 
became  negative,  while  in  the  remaining  35  cases  the 
reaction  is  still  weakly  or  strongly  positive. 

In  Table  24,  I  present  the  data  in  regard  to  the 
time  of  disappearance  of  the  reaction  in  cases  of  dif- 
ferent stages  of  syphilis  after  the  injection  of  "606." 


SERUM  DIAGNOSIS  OF  SYPHILIS.  153 

In  20  cases  where  clinical  improvement  was  very 
slow  the  reaction  was  also  extremely  slow  in  diminu- 
tion and  in  some  cases  it  became  stationary.  In  2 
cases  of  tertiary  syphilis  the  clinical  symptoms  showed 
but  little  improvement,  while  the  reaction  became 
slowly  but  progressively  weaker  within  two  months. 

Six  cases  of  cerebrospinal  syphilis  (Hender- 
son) showed  no  improvement  clinically,  but  the  reac- 
tions were  more  or  less  weakened  within  5  weeks. 

In  2  cases  of  incipient  tabes  (Pedersen)  the 
clinical  and  serological  conditions  improved  in  one 
and  not  in  the  other. 

Ten  relapses  were  observed  in  102  cases.  Most 
of  these  were  cases  of  malignant  or  tertiary  syphilis. 
The  recurrence  of  the  disease  could  always  be  detected 
by  the  return  of  the  reactions,  which  were  diminishing 
until  the  relapse.  Six  relapses  were  reinjected  with 
good  results,  while  one  had  a  second  relapse. 

Among  102  cases  there  were  3  cases  which  showed 
no  effect  upon  the  reaction  as  well  as  upon  the 
symptoms. 

In  4  instances  the  reactions  became  somewhat 
stronger  during  a  few  days  following  the  injection, 
but  their  strengths  were  gradually  reduced  later. 

More  recently,  Craig  conducted  a  very  compre- 
hensive series  of  study  on  the  immediate  effect  of 
salvarsan  treatment  on  the  complement-fixation  test 


154  SERUM  DIAGNOSIS  OF  SYPHILIS. 

( Noguchi  system ) .  The  results  obtained  by  Craig 
are  especially  valuable  because  the  patients  ( soldiers ) 
have  been  and  are  still  being  kept  under  constant  ob- 
servation, thus  affording  him  every  facility  to  make 
the  test  at  whatever  time  chosen.  Under  such  fav- 
orable circumstances  Craig  has  been  examining  700 
soldiers  at  a  period,  and  reported  in  September,  1911, 
an  analysis  of  the  results  on  225  cases  which  had  been 
observed  for  at  least  eight  weeks  since  the  adminis- 
tration of  salvarsan,  and  most  of  them  for  a  longer 
period. 

Of  the  225  cases  reported,  164,  or  72.8  per  cent., 
became  negative,  and  61,  or  27-1  per  cent.,  have  re- 
mained positive.  Of  the  164  cases  which  became  nega- 
tive, 24,  or  14.6  per  cent.,  have  relapsed;  43  have 
remained  negative  for  2  months;  41  for  2y2  months; 
20  for  3  months;  7  for  41/2  months;  11  for  5%  months; 
7  for  6  months,  and  6  for  7  months. 

In  relation  to  the  stage  of  disease  Craig  found 
that  the  reaction  disappears  more  readily  in  primary 
syphilis  (25  out  of  31  cases  ^80.6%)  than  in  sec- 
ondary (100  out  of  135  cases  =  74%),  tertiary  (12 
out  of  22  cases  =  54.9%),  and  latent  period  (27  out 
of  37  cases  =  72.9% ) .  The  number  of  relapses  was 
also  smaller  in  primary  (12%)  than  in  secondary 
(15%),  tertiary  (16.6%),  and  latent  cases  (14%). 

The   rapidity  of  disappearance   of  the  reaction 


SERUM  DIAGNOSIS  OF  SYPHILIS.  155 

varied  according  to  the  stages  of  the  disease.  The 
tertiary  cases  became  negative  more  rapidly  than 
others,  9  of  the  12  becoming  negative  within  2  weeks. 
In  the  secondary  cases  the  reaction  disappeared  most 
frequently  during  the  second,  third  and  fourth  weeks, 
while  the  longest  period  was  8  weeks.  In  the  primary 
cases,  13  of  the  25  became  negative  within  2  weeks, 
and  the  rest  within  5  weeks.  In  latent  cases  the 
majority  became  negative  during  the  second,  third, 
fourth  and  fifth  weeks,  and  no  case  became  negative 
after  6  weeks. 

The  relation  of  the  intensity  of  the  reaction  to  the 
disappearance  of  the  reaction  was  also  analyzed.  Of 
the  119  cases  with  more  than  one  antibody  unit  (com- 
plete inhibition  of  hemolysis)  66.3  per  cent,  became 
negative,  of  which  number  16.4  per  cent,  relapsed.  Of 
71  cases  with  less  than  one  and  more  than  one-half 
antibody  unit  (inhibition  at  least  50% )  80.2  per  cent, 
became  negative,  of  which  12.2  per  cent,  relapsed. 
Of  35  cases  with  less  than  one-half  antibody  unit 
(inhibition  less  than  50%)  80  per  cent,  became  nega- 
tive and  14.2  per  cent,  relapsed.  Nearly  half  of  the 
cases  giving  a  weak  reaction  (less  than  50%  inhibi- 
tion) were  patients  in  the  late  secondary  or  tertiary 
stages  of  the  disease  in  which  the  infection  was  still 
active,  although  they  had  been  treated  vigorously  with 
mercury.     The  one-fifth  of  the  primary  cases  gave 


156 


SERUM  DIAGNOSIS  OF  SYPHILIS. 


also  a  weak  reaction  and  71  per  cent,  of  this  group 
became  negative. 

Among  others  Craig  paid  special  attention  to 
the  relation  of  the  method  of  administration  of  sal- 
varsan  to  the  disappearance  of  the  complement-fixa- 
tion reaction.  The  methods  used  in  the  cases  reported 
were  as  follows:  Injection  of  the  neutral  suspension; 
intramuscular  injection  of  the  alkaline  solution;  intra- 
venous injection,  and  combined  intramuscular  and 
intravenous  injections. 


Became 

negative 

Remained 
positive 

Relapse 

1.  Neutral  suspension 

9 

150 

30 

36 

Per  cent. 
77.7 
78.6 
53.3 

63.8 

Per  cent. 
22.2 
21.3 
46.6 

36.1 

Per  cent. 
71.4 

2.  Alk.  sol.  intramuscular 

6.7 

3.  Intravenous 

37.5 

4.  Combined  intramusc.  and    intra- 
venous  

21.7 

While  the  disappearance  of  the  reaction  was  more 
rapid  in  the  cases  treated  with  the  intravenous  than 
with  the  intramuscular  injection,  yet  the  percentage 
of  the  relapses  was  strikingly  higher  in  the  former. 

The  effect  of  a  previous  mercurial  treatment  was 
also  considered.  It  was  found  that  the  reaction  dis- 
appears more  rapidly  in  the  mercurialized  than  in  un- 
treated cases,  and  the  chances  of  relapse  are  less,  al- 
though relapses  have  occurred  in  patients  previously 
well  treated  with  mercury.    In  the  225  cases  reported, 


SERUM  DIAGNOSIS  OF  SYPHILIS.  157 

110  patients  had  received  no  treatment  previous  to  the 
use  of  salvarsan.  Of  these  82,  or  74.5  per  cent.,  be- 
came negative;  28,  or  25.4  per  cent.,  remained  posi- 
tive, and  10,  or  12.2  per  cent.,  relapsed.  Of  75  cases 
previously  mercurialized,  63,  or  84  per  cent.,  became 
negative;  12,  or  16  per  cent.,  remained  positive,  and 
7,  or  9.3  per  cent.,  relapsed. 

Regarding  the  time  of  relapse  in  24  cases  reported, 
Craig  obtained  the  following  data :  Two  in  4,  four  in 
5,  three  in  6,  four  in  7,  one  in  8,  one  in  9,  one  in  10, 
two  in  11,  two  in  12,  one  in  14,  one  in  16,  one  in  22, 
and  one  in  23  weeks,  after  the  reaction  became  nega- 
tive. A  great  many  of  these  relapsing  cases  had  a 
strong  fixation  reaction  before  the  treatment  with 
salvarsan. 

The  careful  analysis  of  Craig  has  doubtless 
brought  the  serum  reaction  and  the  therapeutic  as 
well  as  prognostic  problems  in  syphilis  to  a  much 
closer  and  clearer  relation  than  has  hitherto  been 
done  by  others.  Craig  and  Nichols  are  inclined  to 
consider  that  the  absence  of  clinical  and  serological 
manifestations  persisting  for  a  period  of  over  one  year 
after  the  treatment  with  salvarsan  indicates  a  cure  of 
the  disease.  In  this  connection  the  writer,  as  also 
mentioned  by  Nichols,  would  suggest  the  use  of  the 
cutaneous  reaction  with  the  luetin  as  another  means 
of  detecting  the  latent  focus  of  the  infection  in  this 


158  SERUM  DIAGNOSIS  OF  SYPHILIS. 

class  of  patients.  According  to  the  writer's  expe- 
rience the  luetin  reaction  reveals  the  latent  syphilis 
in  many  cases  where  the  serum  reaction  or  clinical 
symptoms  are  absent.  The  results  of  the  luetin  test 
on  the  cases  treated  with  salvarsan  and  mercury  and 
where  no  more  clinical  or  serological  signs  of  syphilis 
were  manifest  for  one  year  or  longer  show  that  some 
of  them  still  give  a  distinct  cutaneous  reaction,  while 
others  do  not  respond  to  the  luetin.  It  seems  that 
the  latter  group  of  patients  may  belong  to  the  cured 
cases  of  syphilis  and  the  former  still  harbors  the  in- 
fection in  latency. 


XIII. 

SPECIFIC    COMPLEMENT-FIXATION    IN    SYPHILIS. 

Two  principal  facts,  namely,  the  existence  of  so- 
called  "  antigen  "  in  non-syphilitic  tissues  and  the  oc- 
currence of  the  Wassermann  reaction  in  non-syphilitic 
diseases  (yaws,  leprosy,  malaria,  etc.),  disprove  in- 
directly that  the  Wassermann  reaction  is  caused  by 
the  true  syphilitic  antibodies  and  antigen  (Trepo- 
nema pallidum).  No  one  has  as  yet  conclusively 
separated  the  Wassermann  reaction  from  the  specific 
antibody-antigen  fixation.  The  obstacle  in  settling 
this  point  was  the  lack  of  pure  culture  of  Treponema 
pallidum.  The  pure  culture  of  the  pallidum  furnishes 
us  with  a  material  in  which  we  can  obtain  the  pallidum 
substance  free  from  any  tissue  lipoids  which  may  pro- 
duce the  Wassermann  reaction.  With  a  pure  pal- 
lidum extract  as  antigen  we  can  find  out  whether  a 
given  syphilitic  serum  contains  the  specific  antibody 
or  not. 

After  successful  cultivation  of  Treponema  pal- 
lidum in  1910-1911,  the  writer  immunized  a  number 
of  animals  (rabbits,  sheep)  with  the  pure  pallidum 
extract  for  several  months  and  then  tested  their 
serums  for  the  complement-fixation.  These  were 
tested  simultaneously  with  both  the  pallidum  extract 

159 


160  SERUM  DIAGNOSIS  OF  SYPHILIS. 

and  the  pure  lipoids.  It  was  found  that  these  im- 
mune serums  bind  complement  with  the  pallidum  ex- 
tract, but  not  with  the  lipoids.  On  the  other  hand, 
the  serums  from  rabbits  with  active  experimental 
syphilitic  orchitis  bind  complement  with  the  lipoids, 
but  not  with  the  pallidum  extract.  The  above  find- 
ings show  that  the  immune  serums  contain  the  specie 
antibodies  for  the  pallidum  but  not  for  the  lipoids, 
and  that  in  the  serums  of  syphilitic  rabbits  (active 
stage)  there  is  an  abundance  of  the  lipotropic  sub- 
stance (probably  an  enzyme)  capable  of  rendering 
complement  inactive  (so-called  fixation)  in  the  pres- 
ence of  certain  lipoids,  but  too  little  specific  antibodies 
to  bind  complement  with  the  pallidum  antigen.  This 
completely  demonstrates  the  difference  between  the 
Wassermann  and  the  true  antibody  antigen  reaction 
in  syphilis. 

The  above  results  obtained  in  an  experimental 
syphilis  in  animals  hold  good  also  for  human  syphilis, 
for  the  writer  found  that  the  serums  derfved  from 
primary  and  secondary  cases  of  syphilis  almost  never 
give  a  positive  reaction  with  the  pallidum  antigen, 
and,  if  there  is  any,  it  is  usually  weakly  positive.  The 
same  serums,  as  is  well  known,  usually  give  a  strong 
fixation  with  the  lipoidal  "  antigen  "  (Wassermann 
reaction).  While  there  is  a  striking  absence  of  the 
specific  antibodies  (negative  phase)  during  the  active 


SERUM  DIAGNOSIS  OF  SYPHILIS.  161 

stage  (primary  and  secondary)  of  syphilis,  we  en- 
counter among  the  serums  of  patients  suffering  from 
the  chronic  course  of  syphilis  (tertiary,  latent,  tardive 
congenital)  those  which  contain  varying  quantities  of 
the  specific  antibodies  (positive  phase)  to  be  detected 
with  the  pallidum  antigen.  In  this  latter  class  of  pa- 
tients the  Wassermann  reaction  may  be  positive  in 
some  and  negative  in  others.  It  is  noticed  that  the 
serums  from  patients,  whose  Wassermann  had  dis- 
appeared under  an  energetic  treatment,  give  more 
frequently  a  positive  fixation  with  the  pallidum  anti- 
gen, possibly  due  to  a  more  vigorous  formation  of 
the  specific  antibodies.  Speaking  in  general,  the 
amount  of  the  specific  antibodies  in  human  syphilis 
is,  however,  remarkably  small  and  it  is  rather  infre- 
quent to  obtain  a  complete  exhibition  of  haemolysis 
even  when  the  serum  is  used  in  maximum  quantity. 
On  the  other  hand,  the  immune  serums  (rabbit,  sheep) 
give  a  beautiful  complete  fixation  with  the  pallidum 
extract. 

The  significance  of  a  positive  reaction  with  the 
pallidum  antigen  awaits  the  future  development  for 
its  solution,  yet  it  is  probable  that  a  positive  reaction 
(the  presence  of  the  specific  antibodies)  is  a  favorable 
sign  for  the  patient.  It  is  desirable,  indeed,  to  estab- 
lish a  definite  relationship  between  the  reaction  and 
prognosis.     In  view  of  a  possible  interest  for  the 

12 


162  SERUM  DIAGNOSIS  OF  SYPHILIS. 

serologists  and  clinicians  the  writer  presents  below 
a  brief  technic  for  making  the  fixation  test  with  the 
pallidum  extract. 

The  pallidum  antigen.  Pure  cultures  of  several 
different  strains  of  Treponema  pallidum  growing  for 
different  lengths  of  time  (7,  14,  21,  28,  35,  42  days) 
in  a  special  medium  (consult  the  writer's  publications 
for  the  details)  are  ground  in  a  mill  until  the  pallidum 
is  nearly  disintegrated.  The  thick  emulsion  is  then 
diluted  with  0.9%  salt  solution.  It  is  first  tested  for 
its  anticomplementary  property  in  the  usual  way  of 
titration.  The  amount  of  complement  (guinea-pig's 
fresh  serum)  to  be  used  in  the  titration  is  0.04  c.c.  (or 
0.1  c.c.  of  40%  dilution).  If  the  pallidum  extract 
inhibits  the  action  of  the  complement  in  the  dose  of 
0.2  c.c,  for  example,  one  employs  0.1  c.c.  or  half  of 
the  inhibiting  titre  for  the  fixation  test.  The  antigen 
may  be  preserved  on  ice  with  the  addition  of  0.4% 
phenol.  The  writer  does  not  use  an  emulsion  which 
has  been  standing  over  one  week.  It  is  best  to  test 
its  reliability  by  means  of  an  immune  serum  (rabbit's 
or  sheep's). 

The  patient's  serum  {antibody).  The  serum  of 
the  patient  is  collected  in  the  usual  way  (venepunc- 
ture, etc.).  Before  making  the  test  it  should  be 
heated  to  56°  C.  for  30  minutes  in  a  water-bath.  This 
precaution  is  absolutely  necessary  because  of  the  pro- 


SERUM  DIAGNOSIS  OF  SYPHILIS.  163 

tein  nature  of  the  pallidum  antigen  (see  pages  98- 
101).  For  the  test  0.1-0.2  c.c.  of  the  inactivated 
serum  is  used. 

The  complement,  amboceptor,  and  corpuscle  sus- 
pension. Same  as  given  in  procedures  described  on 
pages  62  and  63. 

llie  te clinic  for  the  fixation  test.  Same  as  indi- 
cated in  the  tables  on  pages  62  and  63.  Special 
attention  is  called  not  to  use  the  fresh  unheated  pa- 
tient's serum  in  this  test,  as  one  is  liable  to  do  so 
v»^hile  following  the  steps  indicated  in  these  proced- 
ures. As  already  stated  above,  0.1-0.2  c.c.  of  the  in- 
activated serum  is  to  be  used,  but  not  one  drop  of 
fresh  serum.  Here  we  are  using  an  antigen  of  pro- 
tein nature,  while  there  (the  lipotropic  or  Wasser- 
mann  reaction)  pure  lipoids  are  employed,  and  hence 
this  distinction. 

COMPLEX  COMPLEMENT-FIXATION  PHENOMENON. 

The  clear  distinction  between  the  Wassermann 
and  the  specific  fixation  reaction  in  syphilis  is  brought 
out  only  through  the  use  of  pure  lipoids  on  one  hand 
and  a  pure  pallidum  extract  on  the  other.  This  is 
no  doubt  a  valuable  distinction,  inasmuch  as  the  for- 
mer indicates  the  activity  of  the  infecting  organism 
(Treponema  pallidum)  and  the  latter  the  immunity 
reaction  on  the  part  of  the  suffering  host.    But  when 


164  SERUM  DIAGNOSIS  OF  SYPHILIS. 

the  complement-fixation  test  is  made  by  means  of 
an  antigen  containing  both  the  active  lipoids  and  the 
pallidum  antigen,  the  result  is  capable  of  being  in- 
terpreted in  two  entirely  diagonal  senses.  It  may 
have  been  a  sign  of  the  activity  of  the  pallidum  or 
that  of  the  host  against  the  pallidum,  or  a  mixture 
of  the  two  phenomena.  The  original  antigen  of  Was- 
sermann,  Neisser  and  Bruck  is  liable  to  bring  about 
this  mixed  phenomena,  because  it  contains  both  the 
lipoids  and  the  pallidum.  As  a  matter  of  fact,  the 
role  of  the  pallidum  in  the  Wassermann  reaction  is 
very  insignificant  in  the  majority  of  instances,  for  the 
reason,  that  whenever  the  reaction  is  very  strong  (as 
in  the  primary  or  secondary  syphilis)  it  is  usually 
caused  by  the  lipotropic  substances  alone  and  not  by 
the  specific  antibodies.  It  may  become  of  some  im- 
portance only  in  the  chronic  cases.  Yet,  if  one  inter- 
prets both  phenomena  in  the  same  sense,  he  is  mis- 
informed. This  confusion  does  not  occur  frequently, 
but  it  should  be  avoided  by  separating  the  two  reac- 
tions by  pure  lipoids  and  pallidum  antigen,  respec- 
tively. The  antigenic  principle  of  the  pallidum  is  in- 
soluble in  alcohol  and  such  an  extract  no  longer  fixes 
complement  either  with  an  immune  serum  or  with 
human  serum  containing  the  specific  antibodies.  From 
this  fact  it  follows  that  an  alcoholic  extract  of  a  foetal 
liver  with  pallidum  contains  no  more  specific  antigen 


SERUM  DIAGNOSIS  OF  SYPHILIS.  165 

and  does  not  differ  from  an  extract  derived  from  a 
normal  liver.  Thus,  the  Wassermann  reaction,  as  is 
understood  today,  is  nothing  but  a  lipotropic  fixation, 
as  practically  every  serologist  is  now  employing  an 
alcoholic  extract  or  a  purified  mass  of  lipoids. 

Recently  W.  H.  Hoffmann  reported  that  by  using 
pure  cultures  of  his  so-called  pallidum  as  antigen,  he 
was  able  to  obtain  the  results  parallel  to  those  obtained 
by  the  use  of  a  syphilitic  liver  extract.  The  spiro- 
ch?eta  which  he  isolated  belongs  beyond  any  dispute 
to  Treponema  microdentium  (Noguchi)  and  differs 
from  the  pallidum  in  producing  a  putrefactive  odor 
and  growing  in  a  serum  agar  (horse  serum  and  agar) 
without  fresh  tissue.  Now  it  is  very  difficult  to  un- 
derstand how  his  culture  happened  to  contain  so  much 
active  lipoids  without  which  the  reaction  could  not 
have  taken  place  in  the  majority  of  syphilitic  serums. 
That  his  culture  is  not  identical  with  the  pallidum  can 
be  easily  shown  by  the  fact,  that  even  the  thickest 
emulsion  of  the  pallidum  derived  from  the  syphilitic 
lesions  of  rabbits'  testicles  or  from  the  pure  cultures 
(Noguchi)  does  not  cause  complement-fixation  with 
most  of  the  active  cases  of  primary  or  secondary 
syphilis,  and  that  even  in  latent  or  tertiary  syphilis  the 
fixation  is  not  so  uniform. 

Before  leaving  this  chapter  the  writer  vdshes  to 
emphasize  that  in  the  later  chronic  stages  of  syphilis 


166  SERUM  DIAGNOSIS  OF  SYPHILIS. 

there  may  be  a  certain  amount  of  specific  antibodies 
and  the  lipotropic  substances,  both  of  which  can  be 
detected  by  an  aqueous  extract  of  a  tissue  rich  in 
j)allidum  and  lipoids,  but  not  in  the  acute  period  of 
the  disease.  In  the  latter  the  fixation  is  caused  al- 
most exclusively  by  the  lipotropic  substances,  and 
the  pallidum  plays  but  a  small  role  in  the  antigen; 
the  specific  antibodies  during  that  period  of  the  dis- 
ease seem  to  be  lacking  or  present  in  a  negligible 
quantity.  It  is  of  advantage  to  carry  out  the  fixation 
test  on  syphilitic  serums  for  the  lipotropic  substances 
(Wassermann  reaction)  and  the  specific  antibodies 
separately,  because  each  has  its  own  significance  for 
the  condition  of  the  patient. 


XIV. 

THE  LUETIN  REACTION. 

Active  or  passive  immunity,  the  state  of  allergy, 
and  numerous  other  phenomena,  which  have  been  so 
commonly  observed  and  profitably  studied  in  certain 
infectious  diseases  since  the  successful  pure  cultiva- 
tion of  their  respective  causative  agents,  are  rather 
imperfectly  understood  in  syphilis.  Soon  after  ob- 
taining pure  cultures  of  Treponema  pallidum,  I  com- 
menced to  study  these  questions  experimentally. 
While  the  results  of  my  experiments  on  the  effects 
of  repeated  injections  of  living  and  killed  pal- 
lida on  experimental  syphilis  in  animals  w\\\  be 
presented  later,  I  wish  to  make  a  brief  report  here 
on  one  of  the  phases  of  study  that  seems  to  me  to  be 
of  interest  not  only  to  those  who  are  engaged  in  ex- 
perimental work  but  also  to  clinicians  at  large; 
namely,  on  a  cutaneous  reaction  in  experimental  and 
human  syphilis. 

Stimulated  by  von  Pirquet's  discovery  of  a  specific 
cutaneous  reaction  for  tuberculosis,  several  investiga- 
tors (Meirowsky,  WolfF-Eisner,  Tedeschi,  Nobl, 
Ciuffo,  Nicolas,  Favre  and  Gauthier,  Neisser  and 
Bruck,  Jadassohn,  and  Fontana)  attempted  to  obtain 
a  specific  reaction  for  syphilis  by  applying  ex- 
tracts of  syphilitic  tissues,  prepared  from  syphilitic 

167 


168  SERUM  DIAGNOSIS  OF  SYPHILIS. 

fetal  liver  or  chancre,  to  the  skin  of  syphilitic 
patients.  Their  results  were,  on  the  whole  and 
in  spite  of  some  encouraging  effects,  contradictory. 
Neisser  and  Bruck,  however,  found  that  a  reac- 
tion similar  to  that  produced  with  syphilitic 
extract  can  be  obtained  also  with  a  concen- 
trated extract  of  normal  liver.  This  peculiarity  of 
the  skin  of  syphilitics  is  ascribed  by  Neisser  to  what 
he  calls  the  state  of  "  Umstimmung "  in  the  later 
stages  of  syphilis.  Neisser,  nevertheless,  expresses 
the  hope  that  the  reaction  may  possibly  be  improved 
by  employing  an  extract  of  the  pallidum  free  from 
tissue  constituents.  This  had  not  been  accomplished 
before.  Pure  cultures  of  Treponema  pallidum  ob- 
viously offer  many  advantages,  since  in  them  we  pos- 
sess not  only  pallida  of  different  ages,  but  also  their 
metabolism  products,  which  doubtless  are  important 
factors  in  establishing  an  allergic  state  in  syphilitic 
subjects. 

THE  PREPARATION  OF  CULTURES  FOR  THE  CUTANEOUS 

REACTION. 

Pure  cultures  of  several  strains  of  the  pallidum 
are  allowed  to  grow  for  periods  of  six,  twelve,  twenty- 
four,  and  fifty  days  at  37°  C.  under  anaerobic  con- 
ditions. One  set  is  cultivated  in  ascitic  fluid  contain- 
ing a  piece  of  sterile  placenta,  and  the  other  in  ascitic 
fluid  agar  also  containing  placenta.     The  lower  por- 


SERUM  DIAGNOSIS  OF  SYPHILIS.  169 

tion  of  each  solid  culture  in  which  a  dense  growth  has 
occurred  is  cut  out  and  the  tissue  removed.  The 
agar  columns  which  contain  innumerable  spirochsetee 
are  then  carefully  ground  in  a  sterile  mortar.  The 
resulting  thick  paste  is  gradually  diluted  by  adding, 
little  by  little,  the  fluid  culture  which  also  contained 
an  enormous  mass  of  the  pure  organisms.  The  di- 
lution is  continued  until  the  emulsion  becomes  per- 
fectly liquid.  The  preparation  is  next  heated  to  60^ 
C.  for  thirty  minutes  in  a  water-bath,  and  then  0.5 
per  cent,  tricresol  is  added.  When  examined  un- 
der the  dark  field  microscope,  numerous  dead  pal- 
lida per  field  may  be  seen.  Cultures  made  from  this 
suspension  remain  sterile,  and  with  them  no  infec- 
tion can  be  produced  in  the  testicles  of  rabbits.  The 
suspension  is  kept  in  a  refrigerator  when  not  in  use. 
I  propose  to  speak  of  this  preparation  under  the 
name  of  luetin. 

In  order  to  ascertain  whether  the  reaction  pro- 
duced with  this  suspension  might  not  be  due  to  the 
introduction  of  the  carbolized  culture  medium  alone, 
it  is  necessary  to  prepare  a  similar  emulsion  with 
uninoculated  media  to  be  used  for  control  purposes. 

METHOD   OF  APPLICATION. 

For  rabbits,  the  hair  on  the  lower  side  of  the  back 
is  shaved  off*  and  one  or  two  injections  of  the  luetin 
and  one  injection  of  the  control  suspension  are  made 


170  SERUM  DIAGNOSIS  OF  SYPHILIS. 

in  diiferent  spots.  The  injections  are  made  intra- 
dermically  with  a  very  fine  needle,  the  amount  in- 
jected being  in  each  case  0.07  of  a  cubic  centimeter. 
For  human  subjects,  the  skin  of  the  upper  arm  is 
selected  as  the  site  for  the  intradermic  injection  of 
the  luetin  and  the  control  suspension.  In  the  earlier 
experiments,  different  spots  on  the  same  upper  arm 
were  used  for  both,  but  now  one  arm  is  used  for 
luetin  and  the  other  for  the  control.  The  amount 
injected  in  each  instance  is  0.07  of  a  cubic  centimeter, 
and  the  skin  is  always  sterilized  with  alcoholic  sub- 
limate solution  before  the  injection. 

THE   CUTANEOUS  REACTION   IN  RABBITS. 

Before  the  skin  test  was  applied  to  human  sub- 
jects, several  series  of  experiments  were  performed 
on  rabbits.  The  tests  may  be  divided  into  five 
groups  according  to  the  treatment  previous  to  the 
cutaneous  tests.  The  first  group  comprised  four  rab- 
bits which  received  within  five  months  twelve  intra- 
venous injections  of  emulsions  prepared  from  syphi- 
litic orchitis  or  rabbits  rich  in  pallida,  the  last  injec- 
tion being  given  two  months  before  the  date  of  ap- 
plication of  the  skin  reaction.  Ten  diiferent  strains 
of  pallidum  were  represented  in  this  series.  The 
second  group  was  composed  of  four  rabbits  which 
had  been  injected  with  similar  emulsions,  the  differ- 


SERUM  DIAGNOSIS  OF  SYPHILIS.  171 

ence  being  that  the  pallidum  had  been  killed  by  heat- 
ing the  emulsions  to  60°  C.  for  thirty  minutes  be- 
fore injection.  The  third  group  included  twelve  rab- 
bits which  were  showing,  four  to  six  weeks  after  inoc- 
ulation, syphilitic  orchitis  experimentally  produced 
with  several  different  pallidum  strains.  The  fourth 
group  consisted  of  four  rabbits  in  which  experimental 
syphilitic  orchitis  had  been  cured  about  four  months 
previously  by  the  intravenous  administration  of  sal- 
varsan.  The  fifth  group  consisted  of  eight  normal 
male  rabbits.  The  intradermic  injections  of  the  lue- 
tin  and  control  emulsions  were  made  on  the  same  day 
to  all  the  rabbits. 

RESULTS  OF  THE  TESTS. 

In  the  control  group,  a  very  slight  erythematous 
condition  appeared  at  the  injection  sites  twenty-four 
hours  after  inoculation,  which  disappeared  within 
forty-eight  hours,  the  skin  resuming  its  normal  ap- 
pearance. The  first  and  second  groups  of  treated 
rabbits  gave  different  reactions.  The  sites  of  injec- 
tion of  the  control  emulsion  became  normal  within 
forty-eight  hours,  while  the  sites  of  injection  of  the 
luetin  became  distinctly  red  and  indurated  after 
forty-eight  hours.  The  point  of  redness  gradually 
spread  and  became  raised  to  the  size  of  a  pea.  This 
condition  persisted  for  four  to  six  days,  when  the  re- 


172  SERUM  DIAGNOSIS  OF  SYPHILIS. 

action  commenced  to  recede.  The  redness  and  in- 
duration disappeared  in  most  animals  within  ten  days. 
In  one  instance,  a  round  sterile  pustule  developed  on 
the  fifth  day  on  the  site  of  each  of  the  two  inocula- 
tions of  the  luetin  (Plate  I).  These  pustules  re- 
sembled variolous  pustules  in  general  appearance 
and  healed  with  crust  formation.  The  third  group 
gave  only  slight  reactions,  which  possibly  were  some- 
what greater  than  in  the  control  rabbits.  But,  as 
a  whole,  they  were  not,  except  in  a  few  instances, 
distinct  enough  to  be  considered  positive.  The  fourth 
group  behaved  like  the  control.  No  constitutional  ef- 
fects were  observed  even  among  the  animals  with 
positive  cutaneous  reactions. 

THE    CUTANEOUS    REACTION    IN    MAN. 

Through  the  courtesy  and  collaboration  of  Dr. 
Martin  Cohen  (Harlem  Hospital,  Randall's  Island 
Asylum,  New  York  Ophthalmic  and  Aural  Insti- 
tute), Dr.  Henderson  (State  Hospital,  Ward's  Is- 
land, New  York),  Dr.  Lapowski  (Good  Samaritan 
Dispensary),  Dr.  McDonald  (Kings  County  Hos- 
pital), Dr.  Orleman-Robinson  ( North- Western 
Clinic,  New  York  Polyclinic) ,  Dr.  PoUitzer  (German 
Hospital),  Dr.  RosanofF  (Kings  Park  State  Hos- 
pital), Dr.  Satenstein  (City  Hospital,  Blackwell's 
Island,  New  York),  Dr.  Schmitter  (Captain  United 


Plate  I. 


'J 


M'^     ^ 


An  actively  immunized  rabbit  (No.  4),  inoculated  on  June  14,  1911.  The  drawing  was  made  on 
the  sixth  day  after  inoculation.  Both  pustules  are  produced  by  the  luetin.  The  control  site  dis- 
appeared. 


SERUM  DIAGNOSIS  OF  SYPHILIS.  173 

States  Army,  at  Fort  Slocum),  Dr.  Schradieck 
(Kings  County  Hospital),  Dr.  Charles  Schwartz 
(California),  Dr.  Smith  (Long  Island  State  Hos- 
pital), Dr.  Strong  (Manhattan  Eye,  Ear,  and 
Throat  Hospital),  Dr.  Swinburn  (Good  Samaritan 
Dispensary),  Dr.  Winfield  (Kings  County  Hos- 
pital), Dr.  Wiseman  (Kings  Park  State  Hospital), 
and  the  Hospital  of  the  Rockefeller  Institute  for 
Medical  Research,  I  was  enabled  to  apply  the  skin 
reaction  to  a  number  of  human  cases. 

The  total  number  of  cases  studied  was  642;  of 
these  315  were  of  syphilitic  nature,  77  of  parasyphi- 
litic  nature,  while  250  represented  various  controls. 

The  emulsions  were  always  injected  intrader- 
mically,  that  is,  in  the  skin  as  superficially  as  possi- 
ble. When  the  injection  is  successfully  carried  out, 
the  epidermic  layer  is  raised  up  sharply  from  the 
cutis,  the  pale  swelling  thus  produced  going  down  in 
from  ten  to  fifteen  minutes. 

DESCRIPTION    OF  THE  REACTIONS. 

Normal  or  Negative  Reactions. — After  applying 
the  emulsions,  both  luetin  and  control,  to  about  fifty 
non-syphilitic  individuals,  I  was  able  to  determine  the 
variations  and  limitations  of  the  reactions  that  follow 
intradermic  administration  in  the  skin  of  non-luetic  in- 
dividuals.    In  the  majority  of  non-luetic  persons 


174  SERUM  DIAGNOSIS  OF  SYPHILIS. 

there  appears,  after  twenty-four  hours,  a  very  small 
erythematous  area  at  and  around  the  point  of  injec- 
tion. No  pain  or  itching  sensation  is  experienced. 
This  reaction  gradually  recedes  within  forty-ei^ht 
hours  and  leaves  no  induration.  In  certain  individ- 
uals, the  reaction  may  reach  a  stage  of  small  papule 
formation  ( about  4x4  mm. )  after  twenty- four  to 
forty-eight  hours,  after  which  and  within  seventy- 
two  hours  it  commences  to  subside.  'No  induration 
is  left  behind,  although  occasionally  slight  yellowish 
pigmentation  may  result  from  mild  ecchymosis. 

Positive  Reactions. — According  to  the  manner 
and  intensity  with  which  the  skin  of  syphilitics  re- 
sponds to  the  introduction  of  the  luetin,  one  may 
distinguish  the  following  varieties  of  effects : 

(A)  Papular  Form  (Plate  II). — ^A  large, 
raised,  reddish,  indurated  papule,  usually  seven  to 
ten  millimeters  in  diameter,  makes  its  appearance 
in  twenty-four  to  forty-eight  hours.  The  papule 
may  be  surrounded  by  a  diffused  zone  of  redness 
and  show  marked  telangiectasis.  The  dimensions 
and  the  degree  of  induration  slowly  increase  during 
the  following  four  or  five  days,  after  which  the  in- 
flammatory processes  begin  to  recede.  The  color  of 
the  papule  gradually  becomes  dark  bluish  red.  The 
induration  disappears  within  two  weeks,  except  in 
certain  instances  in  which  a  trace  of  the  reaction  may 
persist  for  a  longer  period.     This  latter  effect  is 


Plate   II. 


Right.  Left. 

Male,  aged  23  years.  Chancre  four  months  ago,  followed  by  rupio-papular  erup- 
tions, which  have  cleared  up  with  some  scars  in  certain  localities.  Treatment:  three 
intravenous  injections  of  salvarsan  up  to  the  time  of  making  the  luetin  test.  Wasser- 
mann  reaction  weakly  positive.  The  left  arm  shows  the  luetin  reaction  on  its  fifth  day; 
the  right  is  the  control. 


SERUM  DIAGNOSIS  OF  SYPHILIS.  175 

usually  met  with  among  cases  of  secondary  syphilis 
under  regular  mercurial  treatment  in  which  there  are 
no  manifest  lesions  at  the  time  of  making  the  skin 
test.  Congenitally  syphilitic  children  under  the  age 
of  one  year  also  show  this  reaction. 

(B)  Pustular  Form. — The  beginning  and  course 
of  this  reaction  resemble  the  papular  form  until  about 
the  fourth  or  fifth  day,  when  the  inflammatory  pro- 
cesses still  increase  in  intensity.  The  surface  of  the 
indurated,  round  papule  becomes  mildly  edematous, 
and  multiple  miliary  vesicles  occasionally  form 
(Plate  III).  At  the  same  time,  a  beginning  cen- 
tral softening  of  the  papule  can  be  seen.  Within 
the  next  twenty-four  hours,  the  papule  changes  into 
a  vesicle  filled  at  first  with  a  semi-opaque  serum  that 
later  becomes  definitely  purulent  (Plates  III,  IV, 
V).  Soon  after  this,  the  pustule  ruptures  spon- 
taneously or  after  slight  friction  or  pressure.  The 
margin  of  the  broken  pustule  remains  indurated, 
while  the  defect  caused  by  the  escape  of  the  pustular 
content  becomes  quickly  covered  by  a  crust  that  falls 
off  within  a  few  days.  A  small  induration  sometimes 
remains  for  a  few  weeks  or  even  months,  leaving  a 
small  keloid  after  healing.  There  is  a  wide  range  of 
variation  in  the  degree  of  intensity  of  the  reaction 
described  in  different  cases,  as  some  show  rather  small 
pustules,  while  in  others  the  pustule  is  much  larger. 
This  reaction  was  found  almost  constantly  in  cases 


176  SERUM  DIAGNOSIS  OF  SYPHILIS. 

of  tertiary  and  tardive  hereditary  syphilis,  as  well  as 
in  cases  of  secondary  syphilis  which  had  been  treated 
with  salvarsan. 

(C)  Torpid  Form. — In  rare  instances,  the  injec- 
tion sites  fade  away  to  almost  invisible  points  within 
three  or  four  days,  so  that  they  may  be  passed  over  as 
negative  reactions.  But  sometimes  these  spots  sud- 
denly light  up  again  after  ten  days  or  even  longer 
and  progress  to  small  pustular  formation.  The 
course  of  this  pustule  is  similar  to  that  described  for 
the  preceding  form. 

This  type  of  reaction  has  been  observed  in  a  few 
cases  of  primary,  secondary  and  congenital  syphilis; 
quite  frequently  among  the  cases  suffering  from 
syphilitic  affections  of  central  nervous  system. 

Neither  in  syphilitics  nor  in  parasyphilitics  did  a 
marked  constitutional  effect  follow  the  intradermic 
inoculation  of  the  luetin.  In  most  positive  cases,  a 
slight  rise  in  temperature  took  place,  lasting  for  one 
day.  In  several  tertiary  and  tardive  hereditary  cases, 
however,  general  malaise,  loss  of  appetite,  and  diar- 
rhoea were  noted. 

THE  PECULIARITY   OF  THE  SKIN   OF  SYPHILITICS. 

The  skin  of  certain  individuals  suffering  from 
tertiary  syphilis  is  noted  for  its  susceptibility  to  trau- 
matic irritations.    This  tendency  has  been  much  dis- 


Plate  III. 


1^ 


til 


Female,  aged  16  years.  Hereditary  syphilis,  and  interstitial  keratitis  of  six  months' 
standing.  Salvarsan  injected  five  months  ago  without  good  results.  Wassermann 
reaction  positive.  Inoculated  on  June  17,  1911.  The  drawing  was  made  on  the  fifth 
day.  Both  sites  of  the  luetin  injection  show  a  tendency  to  pustulation.  Marked 
telangiectasis. 


SERUM  DIAGNOSIS  OF  SYPHILIS.  177 

cussed  by  dermatologists.  To  account  for  this  phe- 
nomenon, Finger  advanced  the  theory  of  superinfec- 
tion, which  presupposes  that  a  trauma  creates  a  spot 
of  weakened  resistance  in  the  skin  and  that  the  syphi- 
litic virus  wanders  thither  from  a  hidden  focus  in  the 
interior  to  set  up  the  lesion.  On  the  other  hand, 
Neisser  maintains  that  it  is  not  the  spiroch^eta  that 
produces  a  syphilitic  lesion  after  a  slight  trauma,  be- 
cause he  failed  to  prove  the  presence  of  any  spiro- 
chseta  or  infectious  agent  in  the  lesions;  but  he  be- 
lieves that  it  is  due  to  a  pathological  condition  of  the 
skin  itself,  which  he  calls  "  Umstimmung."  Among 
315  cases  of  syphilis,  I  observed  fifteen  cases  (one  sec- 
ondary, twelve  tertiary,  and  two  hereditary)  in  which 
the  sites  of  the  control  emulsion  reacted  quite  in- 
tensely. But  these  reactions  were  usually  less  in- 
tense than  those  produced  by  the  luetin  in  the  same 
persons.  Furthermore,  in  these  cases  the  induration 
remained  much  longer  at  the  sites  of  luetin  injection 
than  at  the  control  sites.  In  view  of  these  results 
and  of  the  additional  fact  that  the  majority  of  cases 
giving  the  positive  skin  reactions  did  not  react  to  the 
control  emulsion,  I  am  of  the  belief  that  the  reactions 
set  up  by  the  intradermic  inoculation  of  luetin  are  not 
due  to  the  abnormal  irritative  condition  of  the  skin 
alone,  but  more  to  a  specific  allergic  condition.  It 
is,  of  course,  possible  that  both  conditions  may  be 

13 


178  SERUM  DIAGNOSIS  OF  SYPHILIS. 

coexistent  in  the  same  patient,  although  the  occur- 
rence of  "Umstimmung "  appears,  from  my  expe- 
rience, to  be  far  less  frequent  than  that  of  allergy. 
In  carrying  out  the  cutaneous  reaction  for  syphilis 
(as  for  any  other  infectious  disease),  it  is  highly  im- 
portant to  provide  adequate  control  observations  to 
determine  whether  the  state  of  "  Umstimmung  "  ex- 
ists only  in  syphilis,  or  whether  it  may  not  also  oc- 
cur in  other  diseases. 

THE  RESULTS  OF  THE  CUTANEOUS  REACTION  IN  MAN. 

In  all,  642  cases,  comprising  i315  syphilitics,  77 
parasyphilitics,  and  250  controls,  have  been  studied. 
The  results  obtained  in  syphilitics  are  summarized  in 
Table  25.  The  table  shows  that  in  cases  of  primary 
and  secondary  syphilis,  which  have  had  either  insuffi- 
cient treatment  or  none  at  all,  no  skin  reaction  oc- 
curred, except  in  a  few  instances.  The  reaction  in 
the  positive  cases  was  always  of  the  indurated  papu- 
lar variety.  In  one  primary  case,  in  which  the  skin 
test  was  applied  before  regular  mercurial  treatment 
was  begun,  a  torpid  reaction  followed  the  adminis- 
tration of  mercury.  On  the  other  hand,  most  of  the 
secondary  cases  which  had  been  mercurialized  before 
the  administration  of  salvarsan,  and  which  remained 
without  symptoms  for  some  months  after  the  salvar- 
san injections,  gave  striking  and  unmistakable 
reactions. 


Plate  IV. 


Male,  aged  29  years.  Chancre  seven  months  ago,  followed  by  secondaries.  Six 
intramuscular  injections  of  salvarsan,  the  last  about  three  months  ago.  Developed  ex- 
udative chorioiditis  six  weeks  ago.  The  right  eye  shows  typical  exudative  chorioiditis; 
the  left  eye  is  free  from  it.  Wassermann  reaction  negative.  Inoculated  on  June  23, 
1911.  The  drawing  was  made  on  the  sixth  day.  Between  the  two  large  luetin  pustules 
there  is  a  faint  trace  of  the  injection  of  the  control   material. 


SERUM  DIAGNOSIS  OF  SYPHILIS. 


179 


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180  SERUM  DIAGNOSIS  OF  SYPHILIS. 

Two  distinct  groups  that  give  a  negative  or  doubt- 
ful reaction  occur  among  the  salvarsan  cases.  One 
consists  of  cases  showing  a  reappearance  of  the  Was- 
sermann  reaction,  but  no  other  symptoms,  and  usually 
gives  doubtful  cutaneous  reactions.  The  other, 
which,  however,  comprised  only  fifteen  cases  treated 
with  salvarsan  and  mercury  that  remained  without 
any  clinical  symptoms  or  Wassermann  reaction  for 
more  than  a  year,  gave  no  cutaneous  reaction. 

To  this  group  also  belong  two  interesting  cases 
that  have  been  under  my  observation  since  Septem- 
ber, 1910.  One  was  treated  for  gumma  of  the  liver 
by  an  intramuscular  injection  of  salvarsan  by  Dr. 
Kakels  at  the  Lebanon  Hospital.  The  large  gumma 
promptly  disappeared,  and  the  patient  has  remained 
without  symptoms  for  two  years.  The  Wasser- 
mann reaction  in  this  patient  disappeared  slowly  but 
completely  in  about  ten  weeks.  The  blood,  tested 
every  two  months,  has  remained  negative.  The  skin 
test  with  luetin,  made  in  August,  1911,  and  January, 
1912,  was  negative.  The  other  case  is  one  of  malig- 
nant syphilis,  refractory  to  mercurial  treatment. 
Three  intramuscular  injections  of  salvarsan  were 
given  in  September  and  November,  1910,  and  in 
January,  1911.  The  Wassermann  reaction  dis- 
appeared soon  after  the  healing  of  the  extensive  and 


Plate   V. 


Left. 


Right. 


Female,  aged  51  years.  Became  infected  twenty  years  ago,  and  was  treated  moderately  by  Lassar  in  Berlin. 
Present  symptoms:  exostosis  and  stenosis  of  lacrimal  canal.  Wassermann  reaction  weakly  positive.  Luetin 
test  on  July  11th.  The  left  arm  shows  two  well  marked  pustules  at  the  sites  of  the  luetin  injection,  while 
the  right  arm  shows  a  faintly  visible  trace  on  the  control  site.  The  photograph  was  taken  on  the  sixth  day. 
(Dr.  S.  Pollitzer  kindly  took  this  photograph  for  me.) 


SERUM  DIAGNOSIS  OF  SYPHILIS.  181 

destructive  lesions,  and  still  remains  negative.  A 
luetin  test  made  in  August,  1911,  and  February, 
1912,  was  negative.  Both  patients  are  now  in  excel- 
lent health.  I  have  introduced  these  cases  to  illustrate 
the  point  that  the  allergic  state  of  the  skin  in  syphilis 
seems  not  to  persist  beyond  a  certain  time  after  the 
probable  complete  eradication  of  the  pallidum  from 
the  body.  This  point  has  been  confirmed  experi- 
mentally by  rabbits  treated  with  salvarsan. 

The  results  in  tertiary  and  late  hereditary  syphilis 
present  a  striking  contrast  to  those  observed  in  the 
earlier  stages  of  the  disease.  The  skin  reacts  in- 
tensely to  luetin  in  these  cases.  The  intensity  of  the 
reaction  in  these  cases  is  probably  increased 
by  the  state  of  "  Umstimmung,"  but  the  constancy 
with  which  it  appears  suffices  to  render  it  a  valuable 
clinical  diagnostic  aid.  It  is  in  this  stage  that  syphilis 
manifests  itself  in  its  most  diverse  and  often  most  ob- 
scure forms.  A  gumma  or  other  tertiary  lesion  on  the 
visible  parts  of  the  body  offers  but  little  diagnostic 
difficulty;  but  it  is  not  always  easy  to  ascertain  the 
luetic  nature  of  lesions  of  the  internal  or  specific  or- 
gans. In  tertiary  cases,  moreover,  the  Wassermann 
reaction  is  frequently  absent,  especially  after  antiluetic 
treatment.  Thus  it  is  important  to  find  that  the  skin 
reaction  is  more  constant  than  the  Wassermann  re- 
action in  tertiary  syphilis.    Two  causes  for  a  negative 


182  SERUM  DIAGNOSIS  OF  SYPHILIS. 

Wassermann  reaction  in  the  tertiary  stage  may  be 
distinguished.  The  first,  which  may  apply  also  to 
latent  syphilis,  consists  of  the  restraining  power  of 
the  body  upon  the  propagation  of  the  pallidum  to- 
gether with  the  neutralization  of  its  injurious  pro- 
ducts through  the  formation  of  antibodies.  The  sec- 
ond cause,  which  also  operates  in  some  primary  and 
secondary  cases,  arises  from  the  inability  of  the  body 
to  respond  to  the  pallidum  stimulus  by  the  formation 
of  the  substances  on  which  the  Wassermann  reaction 
depends. 

The  results  of  the  luetin  reaction  in  syphilis 
of  the  central  nervous  system  appear  less  constant 
than  is  the  Wassermann  reaction,  as  long  as  the  treat- 
ment remains  ineiFective,  but,  judged  from  my  later 
observations,  the  luetin  reaction  develops  in  almost 
every  case  which  had  been  treated  energetically.  In 
these  well-treated  cases  the  Wassermann  reaction 
in  the  blood  usually  disappears.  The  cases  of  latent 
syphilis  reported  here  consist  chiefly  of  parents  of  the 
hereditary  syphilitic  cases  reported  in  Table  25.  Most 
of  them  became  infected  many  years  before  and  have 
remained  without  symptoms.  Many  of  the  mothers 
have,  however,  suffered  miscarriages  previous  to  or 
after  the  birth  of  their  syphilitic  children. 

The  controls  include  50  non-syphilitic  children 
between  the  ages  of  two  and  eighteen  years,  and  200 


SERUM  DIAGNOSIS  OF  SYPHILIS.  183 

adults  suiFering  from  various  diseases  of  non-syphi- 
litic nature.  These  include  tuberculosis,  leprosy, 
pneumonia,  typhoid  fever,  psoriasis,  malaria,  alco- 
holic psychosis,  dementia  prsecox,  gonorrhea,  chan- 
croid, brain  tumor,  eczema,  epithelioma,  carcinoma, 
etc.    In  none  was  a  positive  luetin  reaction  obtained. 

The  results  thus  far  obtained  in  parasyphilitic 
cases  are  unsatisfactory  and  call  for  further  study 
with  more  active  preparations  of  the  luetin.  Seventy- 
two  cases  of  general  paralysis  and  five  of  tabes  were 
studied.  Of  the  seventy-two  cases  of  general  paraly- 
sis, forty-five  reacted  positively.  In  a  few  instances 
pustules  were  formed,  but  twenty-seven  gave  no  re- 
action. Of  five  cases  of  tabes,  three  reacted  posi- 
tively. Among  thirty-five  controls  of  this  series,  con- 
sisting of  fifteen  cases  of  dementia  precox,  six  of  al- 
coholic psychosis,  and  fourteen  of  other  forms  of 
psychoses,  four  reacted  positively,  and  these  were 
cases  of  dementia  prsecox.  These  patients  were 
adults,  and  two  of  them  gave  positive  Wassermann 
reactions.  I  incline,  therefore,  to  the  opinion  that 
they  had  suiFered  either  from  congenital  or  acquired 
syphilis. 

A  large  series  of  dermatological  cases  were 
studied  by  Orleman-Robinson,  whose  results  are 
quoted  below: 


184 


SERUM  DIAGNOSIS  OF  SYPHILIS. 


Table.  26. 


u 

a 

3 

Symptoms 
Present 

Symptoms 
Absent 

Total 

.2 

3 

a 
a 

03 

a 
1 

CI 

a 
g 

a 

L 

W 

Primary  syphilis. . . . 
Secondary  syphilis .  . 
Tertiary  syphilis .... 

Latent  syphilis 

Congenital  syphilis .  . 

4 
35 
17 

5 

2 

[Positive  reaction 
(Negative  reaction 

[Positive  reaction 
(Negative  reaction 

[Positive  reaction 
(Negative  reaction 

[Positive  reaction 
(Negative  reaction 

[Positive  reaction 
1  Negative  reaction 

0 
4 

20* 
15t 

13 
0 

2 
0 

1 
0 

3 

1 

25 1 
10 

8 
5 

1 
1 

1 
0 

4 
0 

3 
0 

1 
0 

2 
2 

0 
3 

1 
0 

+  0 

-  4 

+20 
-15 

+  17 

-  0 

+  5 

-  0 

+  2 

-  0 

3 
1 

25 
10 

10 

7 

1 

4 

2 
0 

*  Under  regular  treatment,     f  No  or  slight  treatment. 
X  The  majority  only  slightly  treated. 


TABLE  27. 
Control  Cases 


Number  of 
Cases 


Acne  vulgaris 9 

Alopecia  areata 3 

Bromide  eruption 1 

Carcinoma 6 

Darier's  disease 1 

Eczema  (various  forms) 15 

Erythema  multiforme 4 

Erythema  toxicum 3 

Herpes  zoster 5 

Ichthyosis 2 

Keloid 2 

Lupus  erythematosus 6 

MoUuscum  contagiosum 1 


Luetin  Reaction 
Positive  Negative 

1*  8 

3 

1 

6 

1 

15 

4 

3 

5 

2 

2 

6 

1 


*  A  woman  of  40  years  of  age  with  a  questionable  history,  admits  two  mis- 
carriages and  is  a  chronic  alcoholic  subject.  The  Wassermann  reaction  could  not 
be  performed,  as  she  objected,  and  would  have  been  of  no  avail  on  account  of 
her  constant  usage  of  alcohol.     Syphilis  cannot  be  excluded  from  this  case. 


SERUM  DIAGNOSIS  OF  SYPHILIS. 


185 


TABLE  27,  Control  Cases — Continued 


Number  ot 
Cases 


Pos; 


Luetin  Reaction 


tive 


Pityriasis  rosea 2 

Psoriasis 14 

Sarcoma  (Kaposi) 1 

Scabies 10 

Sycosis 3 

Tinea  versicolor 2 

Trichophytosis 4 

Tuberculosis  pulmonalis 12 

Xanthoma 2 


Negative 

2 
14 

1 
10 

3 

2 

4 
12 

2 


108 


107 


Nobl  and  Fluss  examined  100  dermatological 
cases  at  Wiener  allgemeine  Poliklinik,  and  made  the 
following  preliminary  report: 


Luetin  reaction 

Primary 
stage 

Secondary 
stage 

Gum- 
matous 
stage 

Late 
latency 

Para- 
syphilis 

Hereditary 
syphilis 

Controls 

Marked 

Weak 

None 

2 
1 
4 

21 
15 
26 

1 

9 
3 

4 

2 

1 
1 
1 

3* 
3* 

_ 

Total.... 

7 

62 

1 

16 

2 

3 

9 

*  4  out  of  the  8  control  cases  gave  positive  Wassermann,  one  case  not  tested. 

These  authors  regret  not  having  had  larger  num- 
bers of  tertiary  and  latent  cases  in  which  the  luetin  re- 
action is  expected  to  come  out  more  constantly  posi- 
tive. They  state  also  that  from  those  control  cases 
which  responded  to  the  luetin  test  a  possibility  of  ex- 
isting luetic  infection  is  hard  to  rule  out,  and  there 
were,  indeed,  4  cases  giving  positive  Wassermann  reac- 
tion among  the  total  8  ( 1  case  not  tested  for  the  serum 
reaction)   examined.     It  will  be  seen  that  the  luetin 


186 


SERUM  DIAGNOSIS  OF  SYPHILIS. 


reaction  was  negative  in  three  out  of  the  four  Was- 
sermann  negative  cases. 

Martin  Cohen  applied  the  luetin  test  to  ophthal- 
mological  cases,  and  obtained  the  following  results: 

Table  28. 


Name  of  disease 


1.  Interstitial  keratitis 

2.  Kerato-iritis 

3.  Keratitis  marg.  profunda 

4.  Keratitis  neuro-paraiytica 

5.  Keratitis  disciformis 

6.  Acute  iritis 

7.  Irido-cyclitis  (acute) 

8.  Irido-choroiditis 

9.  Choroiditis  exud.  (acute) 

10.  Iritis  serosa 

11.  Scleritis 

12.  Optic  neuritis 

13.  Opt.  atrophy   (prim.  3,  sec.  2)  . .  . 

14.  Chorio-retinitis 

15.  Retinitis  proliferans 

16.  Detachm.  of  retina 

17.  Detachm.  of  retina  (traumatic) .  . 

18.  Retinitis  prolif.  (traumatic) 

19.  Macular  choroiditis 

20.  Abducens  paralysis 

21.  Ophthalmopl.  externa 

22.  Thrombosis,  cent.  art.  inf.  br. .  .  . 

23.  Anisocoria 

24.  Stenosis  lachr.  canal 

25.  Puis,  exophthalmos  (idiop.) 

26.  Orbital  tumor 


20 
3 
1 
1 
1 
4 
3 
1 
1 
1 
1 
3 
5 
1 
2 
1 
1 
1 
1 
2 
1 
1 
1 
1 
1 
1 


60 


•a -5 

+   - 


12    8 


24  36 


12 
1 


24 


+    - 


+    - 


11    9 

2    1 

1 
1 
1 

4 

2    1 

1 

1 
1 
1 

3 

4    1 

1* 
2* 
1* 
1 
1 
1 

2 

1 
1 

1 

1 

1 


36      28  32 


36  24 


■  T.  B.  C.  test  + 


+  =  positive,         —  =  negative. 


SERUM  DIAGNOSIS  OF  SYPHILIS.  187 

I.  Cutan.  test  +,  clin.  evid.  +,  Wasserm.  +,  12  cases 
II.  Cutan.  test  — ,  clin.  evid.  — ,  Wasserm.  — ,  16  cases 

28  cases 
I.  Cutan.  test  +,  din.  evid.  — ,  Wasserm.  — ,    3  cases* 
II.  Cutan.  test  — ,  clin.  evid.  +,  Wasserm.  +,  11  casesf 

14  cases 
I.  Cutan.  test  +,  din.  evid.  +.  Wasserm.  — ,    2  cases 
IT.  Cutan.  test  -{-,  clin.  evid.  — ,  Wasserm.  +.    6  cases 

III.  Cutan.  test  — ,  clin.  evid.  — ,  Wasserm.  +,    7  casesj 

IV.  Cutan.  test  — ,  clin.  evid.  +,  Wasserm.  — ,    3  cases || 

18  cases 


60  cases 


*  These  3  cases  may  belong  to  latent  syphilis  in  which  no  clinical  or  sero- 
logical manifestations  are  present. 

t  These  1 1  cases  belong  to  untreated  or  only  slightly  treated  early  syphilis. 

t  These  7  cases  may  belong  to  precose  latent  stage  where  the  clinical  symp- 
toms had  disappeared,  but  the  luetin  reaction  has  not  yet  developed. 

II  These  3  cases  belong  to  an  anomalous  type  of  patients  in  whom  the  Wasser- 
mann  develops  very  slowly  or  may  altogether  remain  missing.  The  luetin 
reaction  has  not  developed  yet. 

The  following  cases  are  cited  here  from  his  ob- 
servations as  they  illustrate  the  practical  value  of  the 
luetin  reaction  in  this  class  of  patients. 

1.  Orbital  Tumor. — A  married  woman,  31  years  of  age,  exhibited 
unilateral  exophthalmos,  orbital  periostitis,  ocular  paralysis  and  keratitis 
lagophthalmos.  Antiluetic  treatment  had  resulted  in  no  improvement. 
The  Wassermann  was  negative.  The  luetin  test  was  negative.  Patho- 
logical examination  of  the  specimen  following  the  Kronlein  operation 
for  orbital  tumor  showed  it  to  be  a  fibro-endothelioma. 

2.  Case  of  Pulsating  Exophthalmos,  Bilateral.— A  married  man,  43 
years  of  age,  whose  wife  had  had  three  miscarriages,  exhibited  eleven 
months  ago  a  protrusion  of  the  right  eyeball;  five  weeks  later  the 
left  eye  also  began  to  protrude.  There  were  also  paralysis  of  both 
external  recti  and  anaesthesia  of  both  cornese.  Pupillary  reactions 
normal.  Visual  fields  normeil.  Vision  markedly  diminished,  more  on 
the  right  side  than  on  the  left.  Veins  of  both  upper  lids  congested, 
also  the  veins  of  both  ocular  conjunctivag.  Ophthalmoscopic  examination 
showed  dilatation  and  tortuosity  of  the  retinal  veins  on  the  left  side, 
but  the  right  fundus  was  normal.  A  distinct  bruit  was  heard  over  the 
left  eyeball  and  left  temple.  Wassermann  and  cutaneous  reactions 
both  positive. 


188  SERUM  DIAGNOSIS  OF  SYPHILIS. 

Four  months  before  the  patient  was  first  seen  he  had  begun  to 
complain  of  roaring  and  buzzing  noises  in  the  head  and  of  severe 
frontal  headaches;  but  there  were  no  other  symptoms.  No  serious 
previous    illness    or    trauma.      The    patient    denied    syphilis. 

After  treatment  with  inunctions  and  grey  powder  for  two  months 
the  exophthalmos  in  the  right  eye  disappeared  entirely  and  improved 
somewhat  in  the  left  eye;  the  paralysis  of  the  external  recti  and  the 
corneal  anaesthesia  disappeared  on  both  sides;  the  venous  engorgement 
in  the  upper  lids  disappeared  on  the  right  side  and  diminished  on  the 
left,  but  the  congestion  of  both  ocular  conjunctivae  persisted.  Vision 
improved  from  20-200  to  20-40  in  the  right  eye,  and  from  20-50  to  30-30 
in  the  left  eye.  The  symptoms,  viz.,  the  buzzing  and  roaring,  dis- 
appeared entirely  and  the  general  condition  improved  markedly.  The 
Wassermann  and  luetin  tests  are  still  both  positive. 

3.  Abducens  Paralysis. — A  patient,  40  years  of  age,  acquired  syphilis 
three  years  ago,  and  has  been  energetically  treated  with  injections 
of  salicylate  of  mercury  since  then.  Diplopia  developed  three  months 
ago.  Wassermann  negative.  Cutaneous  reaction  positive.  Eye  con- 
dition has  improved  greatly  since  salvarsan  was  administered  and 
mercury   readministered. 

4.  Acute  Iridocyclitis,  Unilateral. — A  man,  29  years  of  age,  had 
a  chancre  seven  months  ago,  followed  by  secondary  manifestations. 
Treated  with  six  intramuscular  injections  of  salvarsan,  each  0.3  gm. 
Despite  the  treatment  eye  symptoms  developed  two  months  ago.  Wasser- 
mann repeatedly  negative,  luetin  reaction  strongly  positive. 

On  several  occasions  during  the  first  part  of  1912, 
I  had  the  opportunity  of  demonstrating  the  luetin 
reaction  before  different  medical  societies.  On  these 
occasions  the  patients  were  placed  at  my  disposal  at 
whatever  place  the  meetings  took  place.  The  observa- 
tions on  the  results  of  the  tests  were  absolutely  objec- 
tive, being  made  by  all  those  who  happened  to  be 
present  at  the  time  of  demonstrations.  I  thought  it  of 
interest  to  the  readers  to  see  how  these  demonstrations 
turned  out.  Below  will  be  presented  the  results 
obtained  at  Chicago  and  St.  Louis. 


SERUM  DIAGNOSIS  OF  SYPHILIS.  189 

I.     Chicago  Medical  Society  Series. 

Case  1. — N.  M.,  American  woman,  aged  39,  clinic  patient  of  Drs.  Baum 
and  Corbus.  Present  condition:  Early  secondary  lues.  Treatment: 
Mercury  rubbings.  Wassermann  reaction  not  taken.  Luetin  mild 
positive.    Mar.    14,    1912. 

Case  2.— F.  B.,  white  man,  aged  44,  clinic  patient  of  Drs.  Baum  and 
Corbus.  Present  condition:  Latent  secondaries.  Chancre  in 
October,  1910.    Treatment:   Mercury  rubbings  and  internal.  Wasser- 

^      mann  positive.     Luetin  papular  distinct    (positive).  Mar.  14,   1912. 

Case  3. — J.  D.,  American  man,  aged  34,  clinic  patient  of  Drs.  Baum 
and  Corbus.  Present  condition:  Latent  secondaries.  Infected  two 
years  ago.  Treatment:  Mercury  rubbings.  Salvarsan  injected  in  oil 
suspension  0.6  gm.  Wassermann  positive.  Luetin  mild  positive. 
Mar.    14,    1912. 

Case  4. — S.  B.,  American  man,  aged  26,  clinic  patient  of  Drs.  Baum 
and  Corbus.  Present  condition:  Recurrent  secondaries.  Treatment: 
Mercury  rubbings  intermittent.  Wassermann  positive.  Luetin  posi- 
tive. Mar.  14,  1912. 

Case  5. — ^W.  H.  W.,  American  man,  aged  23,  clinic  patient  of  Drs.  Baum 
and  Corbus.  Present  condition:  Marked  secondaries.  Treatment: 
Salvarsan  in  oil  suspension,  February,  1912.  Mercury  intermittent. 
Wassermann  not  made.     Luetin  positive.  Mar.   14,   1912. 

Case  6. — L.  J.,  colored  man,  aged  23,  clinic  patient  of  Drs.  Baum  and 
Corbus.  Present  condition:  Secondaries.  Eighteen  months  since 
treatment.  Treatment:  Intravenous  injection  of  salvarsan  eight 
months  ago.  Mercury  rubbings.  Wassermann  positive.  Luetin 
suppuration  on  both  arms.  No  difference  between  control  and 
luetin  (Umstimmung?),  Mar.  14,  1912. 

Case  7. — C.  K.,  German  man,  aged  22,  clinic  patient  of  Drs.  Baum 
and  Corbus.  Present  condition:  Secondary  period.  No  symptoms. 
Chancre  four  years  ago.  Treatment:  Mercury  rubbings.  Salvarsan — 
intravenous  injection  May  20,  1911;  June  7,  1911;  October,  1911; 
November,  1911.  Wassermann  not  taken.  Luetin  papular  reaction 
distinct   positive,    Mar.    14,   1912. 

Case  8. — H.  K.,  American  man,  aged  46,  clinic  patient  of  Drs.  Baum 
and  Corbus.  Present  condition:  Cerebral  gumma.  Treatment: 
Mercury  intermittent.  Wassermann  positive.  Luetin  large  full 
indurated  papule.     Typical  positive  reaction.  Mar.  14,  1912. 

Case  9. — J.  S.,  colored  man,  aged  34,  clinic  patient  of  Drs.  Baum  and 
Corbus.  Present  condition:  Probable  tertiary.  No  history.  Treat- 
ment: Internal  about  one  year.  Wassermann  positive.  Luetin 
positive  with  Umstimmung,  Mar.  14,  1912. 


190  SERUM  DIAGNOSIS  OF  SYPHILIS. 

Case  10. — A.  M.,  American  woirian,  aged  21,  clinic  patient  of  Dr. 
Stein.  Present  condition:  Congenital  lues.  Treatment:  None  at 
any  time.     Wassermann  negative.     Luetin  positive,   Mar.    14,   1912. 

Case  11. — C.  H.,  American  man,  aged  16,  clinic  patient  of  Drs.  Baum 
and  Corbus.  Present  condition:  Congenital  lues.  Treatment: 
Mercury  intermittent.  Wassermann  positive.  Luetin  positive. 
Slight   Umstimmung,   Mar.    14,   1912. 

Case  12. — ^W.  J.  H.,  American  man,  aged  42,  clinic  patient  of  Dr. 
Grinker.  Present  condition:  Paresis.  Probably  infected  twenty 
years  ago.  Treatment:  Neglected.  Salvarsan  injected  intravenously 
by  Dr.  Corbus  Dec.  1,  1911,  and  Feb.  5,  1912.  Vigorous  mercury 
rubbings  between  salvarsan  injections.  Wassermann  positive,  Nov. 
28,  1911.  Luetin  positive,  Mar.  14,  1912. 

Case  13. — Control  case.  S.  C,  colored  man,  aged  37,  clinic  patient  of 
Drs.  Baum  and  Corbus.  Present  condition:  No  history  of  initial 
lesion.  No  secondaries.  Believed  to  have  been  infected  twenty 
years  ago.  Treatment:  Mercury  intermittent.  Wassermann 
negative.     Luetin   negative.   Mar.   14,   1912. 

Case  14. — C.  D.,  American  man,  aged  26,  private  patient  of  Dr. 
Corbus,  treated  after  the  biologic  method.  Present  condition: 
Probably  cured.  Tonsillar  chancre,  November,  1910.  Treatment: 
Salvarsan  injected  intramuscularly,  Nov,  7,  1910,  and  Dec.  15,  1910. 
Wassermann  positive,  Nov.  28,  1910;  negative,  Jan.  23,  1911; 
negative.  May  9,  1911;  negative,  March  12,  1912.  Luetin  negative, 
Mar.    14,    1912. 

Case  15. — F.  S.,  American  man,  aged  22,  private  patient  of  Dr.  Corbus, 
treated  after  the  biologic  method.  Ptesent  condition:  Eariy 
secondaries.  Last  symptoms,  Sept.  4,  1911.  Treatment:  injected 
salvarsan  in  oil  suspension,  Sept.  4,  1911;  intravenously,  Feb.  6, 
1912.  Vigorous  mercury  rubbings  between  salvarsan  injections. 
Wassermann  negative,  Jan.  16,  1912.    Luetin  positive.  Mar.  14,  1912. 

Case  16. — C.  L.,  American  man,  aged  38,  private  patient  of  Dr.  Corbus, 
treated  after  the  biologic  method.  Present  condition  incipient 
tabes.  Infected  eleven  years  ago.  Treatment:  Salvarsan  injected 
intramuscularly,  Jan.  9,  1911,  and  March  5,  1911;  intravenously, 
Oct.  6,  1911,  and  Jan.  13,  1912.  Vigorous  mercury  rubbings  between 
injections.  Wassermann  positive,  Nov.  28,  1910;  positive,  Feb.  23, 
1911;  negative,  July  7,  1911;  negative,  Oct.  6,  1911;  negative, 
Jan.   9,   1912.     Luetin   negative.   Mar.    14,    1912. 

Case  17. — M.  A.,  American  man,  aged  36,  private  patient  of  Dr.  Corbus, 
treated  after  the  biologic  method.     Present  condition:    Secondaries. 


SERUM  DIAGNOSIS  OF  SYPHILIS. 


191 


Last  appearance  of  palmar  syphilis,  seven  months  ago.  Treatment: 
Injected  salvarsan  intramuscularly,  Jan.  38,  1911;  intravenously, 
Mar.  18,  1911;  intramuscularly,  August,  1911.  Vigorous  mercury 
rubbings  between  salvarsan  injections.  Wassermann  negative,  Aug. 
22,   1911;   negative,  Jan.   16,   1912.     Luetin  positive.  Mar.   14,   1912. 

Case  18. — P.  S.,  American  man,  aged  22,  private  patient  of  Dr.  Corbus, 
treated  after  the  biologic  method.  Present  condition:  Cured. 
Primary  lesion,  Jan.  21,  1909.  Treatment:  Vigorous  mercury 
rubbings.  Cure  obtained  before  salvarsan  was  administered.  In- 
jected salvarsan  intramuscularly,  Jan.  17,  1911.  Wassermann 
negative,  Dec.  10,  1910;  Feb.  15,  1911;  April  15,  1911;  Aug.  1, 
1911,  and  Dec.  15,  1911.     Luetin  negative.  Mar.   14,  1912. 

Case  19. — O.  J.  G.,  American  man,  aged  31,  private  patient  of  Dr. 
Corbus,  treated  after  the  biologic  method.  Present  condition: 
Latent  secondaries.  Chancre,  September,  1905.  Last  symptoms, 
Oct.  20,  1910.  Treatment:  Neglected  at  first.  Injected  sal- 
varsan intramuscularly,  Oct.  20,  1910,  and  Dec.  11,  1910;  intra- 
venously, Sept.  2,  1911,  and  March  9,  1912.  Vigorous  mercury 
rubbings  between  salvarsan  injections.  Wassermann  positive,  Nov. 
11,  1910;  negative,  Jan.  1,  1910;  positive,  March  8,  1912.  Luetin 
positive.  Mar.  14,  1912. 

Case  20. — A.  G.,  American  man,  aged  20,  private  patient  of  Dr. 
Corbus,  treated  after  the  biologic  method.  Present  condition: 
Secondaries.  Infected  Jan.  1,  1911.  Last  symptoms  May  6,  1911. 
Treatment:  Internal.  Salvarsan  injected  in  oil  suspension,  0.6  gm.. 
May  6,  1911;  intravenously,  0.4  gm.,  Feb.  24,  1911.  Vigorous 
mercury  rubbings  between  injections  of  salvarsan.  Wassermann 
positive,  April  20,  1911,  and  March  4,  1912.  Luetin  positive.  Mar. 
14,   1912. 


II.     St.  Louis  Medical  Society  Series. 


Luetin 
St.  Louis  Hospital. 

1.  Conner,   infection   6   months, 

one  month  ago  rupial  erup- 
tion, almost  well,  Hg  med..     Negative 

2.  Willard,  2  months  ago,  ini- 
tial lesion,  3  weeks  ago, 
secondary,  3  weeks  ago,  0.6 

gm.  salvarsan    Positive 


Wassermann 


Three  weeks  ago 
positive 


Positive 


192 


SERUM  DIAGNOSIS  OF  SYPHILIS. 


Luetin 

3.  Hoffman,  three  penis  lesions, 
probably    chancroidal Negative 

4.  Morelock,     initial     lesion     6 

weeks  ago Negative 

5.  Smith,  mac.  pap.  erup.  ap- 
pearing 3  weeks  ago,  ini- 
tial lesion  42  days  ago Negative 

6.  Dunroy,    12  years    ago    initial 

lesion,    sores    on    lower    lip, 

lingua  geogr Positive 

7.  Wagner,     trauma     of     nose 

simulating  saddle  nose.     No 

history   of  lues Negative 

8.  Murphy,   6  years   ago   initial 

lesion,  3  months  ago  ulcera- 
tive lesions  on  legs,  3  weeks 
ago  salvarsan  intramusc.  . . .     Positive 

9.  Wilson,  9  years  old  infection, 

saddle  nose,  etc Positive 

10.  Pender  (control),  gonor. 
arthritis Negative 

11.  Brown       (control),       gonor. 

acuta Negative 

Alexian    Bros.    Hospital    Cases. 

12.  McLaughlin,  diagnosis  ?   Negative 

13.  Carstaphan,  general  paresis. . .     Negative 

14.  Shaffner,  C.  sp.  lues Positive 

15.  Mack,  definite  history  lues.  .  .     Positive 

16.  Baby,  Dr.  Lipman's  case,  6 
weeks  old,  congenital  syphilis 
(case  has  yielded  to  Hg 
medication) Negative 


Wassermarm 
Negative 

Positive 
Positive 
Positive 
Negative 

Negative 
Negative 
Negative 
Negative 

Negative 

Positive  blood  and 

c.  s.  fld. 
Positive 

Positive 


Blood   of  mother 
negative 


SERUM  DIAGNOSIS  OF  SYPHILIS. 


193 


Luetin  Wassermann 

17  Pearson  (Dr.  Schuchat),  pri- 
mary lesion,  3  weeks  old, 
classical  Hunterian  chancre, 
spirochaeta  in  dark  field ....     Negative  Positive 

18.  Lewis,  suspicious  sore  on 
penis,    no     spirochaeta     found 

in  dark  field Negative  Negative 

St.  Louis  City  Hospital  Medical 
Causes : 

19.  Stebbins,  liver  syphilis,  ter- 
tiary       Positive  Positive 

( Umstimmung) 

20.  Franklin,  gall-bladder  ? Negative  Negative 

21.  Burke,  surgical    (control)  .  . .     Negative  Negative 

22.  Brown,  diabetes  bronze,  liver 

cirrhosis Negative  Negative 

23.  Broadwell,    tertiary    syphilis 

(Dr.  Englebach)    Positive  Negative 

24.  Barrows    (Dr.  Richeter),  an- 

eurism aortse    Positive  Negative 

( Umstimmung) 

25.  McLaren,  aneurism  aortae ....     Positive  Positive 

(Umstimmung) 

26.  Carmody  (control) Negative  Negative 

27.  Coff  (control) Negative  Negative 

28.  Broskow    (control)     Negative  Negative 

29.  DeWorth  (Dr.  Meisenbach), 
5  years'  treatment,  3  Wasser- 

manns  by  3  different  men 
negative.  This  test  applied 
2  years  after  treatment 
stopped   Negative  Negative 

30.  Carman,  latent,  3  years  after 
infection,  sores  in  mouth,  sal- 
varsan    administered.      Test 

to  be  repeated Positive  Positive 

31.  Crohn  (Dr.  Suggett),  tertiary 

lesion  nose   Negative  Positive 

14 


194 


SERUM  DIAGNOSIS  OF  SYPHILIS. 


Luetin  Wassermann 

32.  Dr.  R.  S.  B.,  old  well-treated 

case,  blood  tested  repeatedly, 

always   negative Negative  Negative 

33.  G.  T.  (Dr.  Harris),  incipient 

tabes    Negative  Positive 

34.  Brankston,    syphilis   4   years, 
very     little    treatment,     sore 

throat,  enlarged  glands,  etc.     Positive  Positive 

35.  Walsh    (Frisco   Hosp.),  man 

unconscious,  aphasia,  etc. 
Test  applied,  but  death  en- 
sued in  24  hours,  no  P.  M. . .      ?  Positive 

36.  Rice,  old  case,  well  treated . .     Negative  Negative 

37.  Rowe,    cong.    case,    supposed 
reinfection,  and  reported  as 

such.  (Drs.  Sugget  &  Graves).    Negative  Negative 

38.  Wolfert,    5    years'    infection, 

very  little  treatment,  liver  up 
to  3d  rib,  3  Wassermanns 
in  6  weeks  negative 


39.  Arnold,  well-treated  case  for 

6  years,  3  Wassermanns  in 
last  2  years,  negative. 

40.  Mrs.  Norris  (Dr.  Graves),  c. 
sp.  lues  (osteomalacia  or 
neurasthenia),  3  blood  tests 
positive.  Husband's  blood 
negative 

41.  Kelly,  mac.  pap.  syph.  severe 

type  1  year  ago,  constitu- 
tional symptoms  pronounced, 
fever,  etc.,  Salvarsan  at  that 
time  followed  by  Hg.  No 
symptoms    now 

42.  Mennie  (Dr.  Chilton),  case 
5  years  old,  palmar  syphilis 
last  year  over  hands  like  a 
gauntlet.  Salvarsan  alone 
cleaned  it  up,  resistant  to  Hg 


Positive  Positive,  after 

(Umstimmung)       KI  for  10  days. 


Negative 


Negative 


Positive  Positive 

(  Umstimmung  ) 


Positive 


Positive 


SERUM  DIAGNOSIS  OF  SYPHILIS.  195 

Luetin  Wassermann 

altogether.      Three   intraven- 
ous injections  in  past  year..     Positive  Negative 

43.  Steyns  (Dr.  Dorsett),  sup- 
posed syphilis,  stricture  rec- 
tum, no   history Negative  Negative 

44.  Ernst,  optic  nerve  atrophy, 
luetic  case  one  and  a  half 
years  old,  salvarsan  in- 
tramusc.  1  year  ago  into  hip. 
One  week  ago  another  sal- 
varsan intravenous  injected 
with  Hg  injections  now  being 

taken Negative  Positive 

The  above  series  of  cases,  though  limited  in  num- 
ber and  varieties,  confirm  practically  all  essential 
points  brought  out  by  different  investigators  that 
the  luetin  is  specific  for  syphilis,  and  occurs  most 
constantly  and  intensely  during  the  period  of 
tertiary  syphilis.  In  primary  or  secondary  stages 
the  luetin  reaction  is  usually  absent  or  very 
mild,  while  it  may  become  more  intense  after  the 
energetic  treatment.  In  congenitally  S3^philitic  in- 
fants it  is  less  marked  than  after  many  years'  dura- 
tion, when  the  reaction  becomes  more  pronounced 
and  constant. 

The  recent  investigations  of  Julian  Wolfsohn  on 
the  luetin  reaction  made  at  the  Johns  Hopkins  Hos- 
pital, in  the  medical  division  under  Professor  L.  F. 
Barker,  are  particularly  interesting  and  valuable,  as 
the  varieties  of  diseases  studied  by  him  were  chiefly 


196 


SERUM  DIAGNOSIS  OF  SYPHILIS. 


internal  where  syphilis  may  play  important  parts  in 
etiologjT-  and  a  specific  diagnostic  test  may  find  its 
practical  usefulness.  The  following  table  shows  the 
results  of  his  study : 

Table  29. 
Secondary  Syphilis. 


Diagnosis 

Treatment 

Luetin 
reaction 

Wassermann 

Remarks 

Secondary   syphilis 
(maculopapular) 

Secondary  syphilis 

"606"  24  hrs. 
before  luetin 
test  given 

"606"  given  48 
hrs.  before  lue- 
tin test 

+ 

+ 

+ 
+ 

Temp.  99.8° 

2 

2 

Tertiary  Syphilis. 


Diagnosis 

Previous  treatment 

Luetin 
reaction 

Wassermann 
reaction 
Blood      C.  S.  F. 

Remarks 

SyphUitic   cirrhosis 

1896-1899      long 

_ 

+ 

Not  done 

— 

of  liver.    Pulmon- 

intervals 

ary   tuberculosis 

Syphilitic  periostitis 

3  months.    Chan- 
cre, secondaries 

+ 

+ 

Not  done 

Large   axillary 
glands 

Syphilitic   periosti- 

None (no  hist.) 

+ 

+ 

Not  done 

- 

tis 

Syphilitic   periosti- 

None (no  hist.) 

+ 

+ 

Not  done 

— 

tis.    Oummata 

Hemiplegia.  Arterio- 

"606" 1  yr.  pre- 

+ 

— 

— 

sclerosis 

viously.        No 
hist. 

Luetic     periostitis. 

None    (no    hist.) 

+ 

+ 

Not  done 

Tender  arm 

Polyarthritis 

Miscarriage. 

5  posi- 
tive 

1  nega- 
tive 

5 

1 

positive 
negative 

SERUM  DIAGNOSIS  OF  SYPHILIS.  197 

Latent  Syphilis. 


Diagnosis 

Previous  antisyphilitic 
treatment 

Luetin 
reaction 

Wassermann 

reaction 

Blood       C.  S.  F. 

Remarks 

Acute  lobar  pneu- 

2 months  (chancre. 

+ 



Not  done 



monia 

1909) 

Typhoid  fever  (?) 

1-2  days  seconda- 
ries, 1900 

+ 

Very  tender 
axillary 
glands  2  wks. 

Pulmonary    tuber- 

1  yr.   constantly 

+ 

- 

Not  done 

- 

culosis 

(chancre,  1911) 

Subacute   appendi- 

None (no  hist.) 

+ 

- 

Not  done 

— 

citis 

Aortic  insufficiency 

None  (no  hist.) 

+ 

— 

Not  done 

— 

Pregnancy 

None  (macerated 
foetus) 

+ 

+ 

Not  done 

— 

Brain  tumor.   Hys- 

6 mos.   (chancre. 

+ 

— 

— 

teria 

1895) 

Latent        syphilis. 

"606"  1911 

+ 

- 

Not  done 

— 

"Basilar   menin- 

(chancre, 1873) 

gitis  " 

Hysteria? 

None  (no  hist.) 

+ 

- 

Not  done 

- 

Rheumatic     fever. 

None  (no  hist.) 

+ 

- 

Not  done 

Nodules    were 

Syphilis  (Wasser- 

very  tender 

mann) 

Chronic  nephritis 

None  (no  hist.) 

+ 

- 

Not  done 

- 

Splenic  anaemia 

None      (chancre, 
1904)    (cauter- 
ized) 

+ 

Tachycardia 
to  100.  Ab- 
dominal pain 
over  enlarged 
spleen 

Posterior  urethritis. 

None  (no  hist.) 

+ 

+ 

Not  done 

- 

Tuberculosis. 

Syphilis   (Wass.) 

Catarrhal  jaundice. 

None  (no  hist.) 

+ 

+ 

Not  done 

- 

Syphilis    (Wass.) 

Hysteria.    Syphilis 

1  year  at  intervals 

+ 

+ 

Not  done 

Bone  pains 

(Wass.) 

and  nausea 
12  hrs.  after 
i  nj  ection 
Temp.  99.8° 

198  SERUM  DIAGNOSIS  OF  SYPHILIS. 

Latent  Syphilis. — Continued. 


Diagnosis 

Previous  antisyphilitic 
treatment 

Luetin 
reaction 

Wassermann 

reaction 

Blood       C.S.F. 

Remarks 

Acute       bronchitis. 

None  (no  hist.) 

+ 

-j-     Not  done 



Syphilis      (Was- 

sermann) 

Pulmonary    tuber- 

None   (no    hist.) 

+ 

—     Not  done 

— 

culosis.  Syphilis? 

(Patient       has 

old  iritis) 

+ 

- 

— 

Carcinoma  of  head 

None.      Chancre, 

+ 

—     Not  done 

- 

of  pancreas 

1872 

Paroxysmal  tachy- 

None (no  hist.) 

+ 

+     Not  done 

- 

cardia.     Syphilis 

(Wass.) 

Pregnancy.    Syphi- 

None (macerated 

+ 

-|-     Not  done 

- 

lis  (Wass.) 

foetus) 

Pregnancy.    Syphi- 

None (macerated 

+ 

—     Not  done 

- 

lis     (Wass.)      in 

foetus) 

child 

Hemiplegia 

No      hist.        No 
treatment 

+ 

—     Not  done 

— 

Carcinoma  of  jaw 

Treatment  1  mo. 
each  year,   for 
10  yrs.  (chancre 
1902) 

+ 

-\-     Not  done 

23  pos- 
itive 

0  neg- 
ative 

8  positive 
15  negative 

SERUM  DIAGNOSIS  OF  SYPHILIS. 


199 


Parasyphilis. 


Diagnosis 

Previous  antisyphilitic 
treatment 

Luetin 
reaction 

Wassermann 
reaction 
Blood       C.S.F. 

Remarks 

Cerebro  spinal 

3  days.  Chancre, 

+ 

+ 

Tachycardia 

syphilis.     Begin- 

1899.     Iritis, 

to  110. 

ning       dementia 

1899 

paralytica 

Myocardial  insuflS- 

3  weeks.  Chancre, 

- 

-|-     Not  done 

- 

ciency.     Aortic 

1887 

insufficiency. 

Syphilis  (Wass.) 

Transverse  myelitis 

3-4     months. 
Chancre,   1902. 

+ 

—     Not  done 

— 

Secondaries 

+ 

— 

Not  done 

Myocardial  insuffi- 

None.     Seconda- 

+ 

—     Not  done 

— 

ciency.     Aortic 

ries,  1911 

insufficiency 

Aneurism     aortic 

None.      Chancre, 

+ 

—     Not  done 

^  — 

arch 

1908 

Myocardial    and 

None  (no  hist.) 

+ 

—     Not  done 

— 

aortic      insuffi- 

ciency 

Myocardial  insuffi- 

None (no  hist.) 

+ 

- 

ciency 

Not  done 

Myocardial  insuffi- 

None (no  hist.) 

+ 

- 

ciency 

Aortic  insufficiency 

1  year.    Chancre, 
1899.  Seconda- 
ries, 1899 

+ 

—     Not  done 

Dementia     paraly- 

Short while.? 

+ 

— 

— 

tica,  C.  S.  Lues 

Chancre 

Aneiu-ism  of  aortic 

None 

+ 

+     Not  done 

— 

arch.  Myocardial 

insufficiency. 

Syphilis  (Wass.) 

Aneurism     of     ab- 

3 mos.  (1890)  (pri- 

+ 

-f     Not  done 

— 

dominal      aorta. 

mary  and  sec- 

Syphilis (Wass.) 

ondaries) 

200 


SERUM  DIAGNOSIS  OF  SYPHILIS. 


Parasyphilis. — Continued. 


Diagnosis 

Previous  antisyphilitic 
treatment 

Luetin 
reaction 

Wassermann 
reaction 
Blood       C.S.F. 

Remarks 

Tabes    dorsalis. 

1     mo.    1897,    3 

+ 

-M911 



Acromegaly. 

doses  of  "606" 

+  1912 

Syphilis  (Wass.) 

1911-1912.  Pri- 
maries and  sec- 
ondaries 

-1912 

Arteriosclerosis. 

3-4  weeks,  1900. 

+ 

-{-     Not  done 

— 

Aortic      insuffi- 

Primaries   and 

ciency.     Syphilis 

secondaries 

(Wass.) 

Myocardial  insuffi- 

1   week    (1889). 

+ 

-f     Not  done 

— 

ciency.        Aortic 

Chancre 

insufficiency. 

Syphilis  (Wass.) 

Myelitis 

None 

+ 

—     Not  done 

- 

Tabes    dorsalis. 

3  months  (1887). 

+ 

— 

— 

Chancre.  Nephri- 

Chancre 

tis 

Aneurism  of  aortic 

1   year,    1892. 

+ 

+     Not  done 

— 

arch 

Chancre 

Tabes  dorsalis 

None  (no  hist.) 

+ 

Fever  to  103°, 
pain      in 
abdomen, 
vomiting 
and    nausea 
beginning  30 
hours     after 
and    lasting 
4  days. 

18  pos- 
itive 

1    neg- 
ative 

10  positive 

1  negative 

1  C.  S.  F. 

positive 

The  foregoing  series  was  accompanied  by  a  series 
of  non-syphilitic  cases.  Of  the  seventy  controls  in 
which  the  patients  were  suffering  from  diseases  other 


SERUM  DIAGNOSIS  OF  SYPHILIS.  201 

ihan  syphilis,  e.g.,  soft  chancre,  mitral  insufficiency, 
myocardial  insufficiency,  cancer  of  the  tonsil,  acute 
rheumatic  fever,  pregnancy  with  still-born  infant, 
senile  palsy,  bronchitis,  brain  tumor,  diabetes  mellitus, 
pleurisy  with  effusion,  pericarditis,  hypophysis  tumor, 
pernicious  anaemia,  epilepsy,  peliosus  rheumatica, 
sarcoma  of  the  ileum,  myelogenous  leukaemia,  car- 
cinoma ventriculi,  thyreoid  cyst,  pulmonary  tubercu- 
losis, chronic  nephritis,  rickets,  chronic  constipation, 
gastric  ulcer,  uraemia,  typhoid  fever,  and  infectious 
arthritis,  etc.,  when  no  history  of  syphilitic  infection 
could  be  obtained  and  where  the  Wassermann  re- 
action was  negative  in  each  case,  no  positive  luetin 
or  control  reactions  were  obtained.  In  two  cases, 
small  non-indurated  pustules  developed  within  3  days 
after  the  injection,  but  these  easily  could  be  distin- 
guished from  the  positive  reactions.  No  constitu- 
tional symptoms  were  complained  of  or  noted  in 
these  cases. 

The  analysis  and  discussion  of  the  above  cases  by 
Wolfsohn  are  quoted  below,  as  they  bring  out  many 
interesting  and  instructive  points  regarding  the  luetin 
reaction  in  its  relation  to  clinical  manifestations  and 
the  Wassermann  reaction. 

Secondary  Syphilis. — Two  cases  of  secondary 
syphilis,  both  in  the  maculo-papular  stage,  were  in- 
oculated. In  each  case  the  Wassermann  reaction  was 
positive  and  each  patient  had  been  given  0.4  gram 


202  SERUM  DIAGNOSIS  OF  SYPHILIS. 

salvarsan  intravenously  24  to  48  hours  before  the 
test  was  given.  Both  tests  were  positive.  In  the 
first  case  the  salvarsan  had  been  administered  only 
24  hours  before,  but  the  reaction  to  the  luetin, 
although  delajT^ed  till  the  fifth  day,  was  definitely 
positive.  No  constitutional  symptoms  or  reaction 
over  the  site  of  injection  of  the  control  emulsion 
( "  I Jmstimmung  " )   were  noted. 

Tertiary  Syphilis. — This  series  includes  six  cases, 
in  which  the  Wassermann  reaction  was  positive  in 
five.  The  sixth  patient  had  had  0.6  gram  of  salvarsan 
one  year  before  admission  and  his  test  proved  nega- 
tive both  for  blood-serum  and  cerebrospinal  fluid  at 
this  time.  The  Wassermann  was  positive  in  1911, 
however.  The  luetin  reaction  in  this  case  was  posi- 
tive. In  the  first  case  of  this  series  the  luetin  re- 
action was  negative  though  the  Wassermann  was 
positive.  This  case  was  not  followed  longer  than  10 
days. 

The  reaction  in  four  cases  of  luetic  periostitis  was 
of  the  violent  variety  and  showed  early  pustule  forma- 
tion and  rather  marked  reaction  on  the  control  side 
( "  Umstimmung  " ) .  Two  of  these  complained  of 
tender  and  painful  arms  and  one  developed  enlarge- 
ment of  the  axillary  glands.  All  these  manifestations 
disappeared  in  the  following  48  hours. 

It  is  interesting  to  note  that,  in  one  case,  two 
weeks  after  subsidence  of  the  local  manifestations 


SERUM  DIAGNOSIS  OF  SYPHILIS.  203 

there  was  a  flare-up  at  the  site  of  the  injection  of 
luetin.  A  pustule  formed  and  rapidly  disappeared, 
leaving  no  other  signs  than  slight  desquamation  and 
pigmentation. 

The  rapidity  with  which  the  reaction  becomes 
manifest,  the  ease  with  which  it  is  interpreted,  to- 
gether with  the  almost  constant  development  of  it, 
in  this  stage  of  the  disease,  makes  it  a  most  valuable 
aid  in  diagnosis.  It  not  only  checks  up  the  Wasser- 
mann  reaction,  but  it  may  be  indeed  supplementary 
to  it,  especially  in  the  treated  cases,  in  which  the 
Wassermann  reaction  not  infrequently  may  be 
negative. 

Parasyphilis.- — If  one  accepts  the  newer  concep- 
tion of  parasyphilis  he  will  include  not  only  tabes 
and  general  paresis,  but  those  cases  which  show  vascu- 
lar changes  of  luetic  origin,  e.g.,  cases  of  syphilitic 
mesaortitis  and  aortic  aneurisms.  Therefore,  in- 
cluded in  this  series  are  nineteen  cases,  seven  of  which 
are  of  central  nervous  system  affection,  and  twelve 
of  cardiovascular  disturbance. 

A  careful  analysis  of  these  cases  shows  the  great 
value  of  the  luetin  test.  Of  the  former  cases  all 
seven  gave  positive  tests,  while  the  Wassermann  test 
was  negative  in  six.^     One  of  these,  a  tabetic,  had  a 

^  Of  course  one  must  keep  in  mind  the  limited  number  of  cases 
that  the  luetin  test  has  been  tried  on;  the  result,  however,  even  in 
this  small  series  has  been  striking. 


204  SERUM  DIAGNOSIS  OF  SYPHILIS. 

positive  Wassermann  reaction  one  year  previously 
and  had  had  in  the  interim  three  doses  of  salvarsan, 
but  the  cerebrospinal  fluid  still  gave  a  positive 
Wassermann  test.  The  luetin  reaction  in  this  case 
was  violent,  the  reaction  over  the  site  of  control  in- 
jection ( "  Umstimmung  " )  being  especially  notice- 
able, as  it  was  also  in  five  of  these  cases. 

Another  tabetic,  who  showed  no  other  signs  than 
diminished  knee-kicks  and  sluggish  pupils,  developed 
remarkable  constitutional  symptoms,  beginning  30 
hours  after  inoculation  and  lasting  four  days.  These 
symptoms  consisted  of  fever  as  high  as  103°,  pain  in 
the  abdomen  resembling  crises,  with  nausea  and 
vomiting.  The  reaction  was  violent  and  "  Umstim- 
mung "  was  present.  The  Wassermann  reaction  was 
negative,  both  for  blood-serum  and  cerebrospinal 
fluid  in  this  case.  This  patient  had  never  received 
treatment  previous  to  this  time. 

Of  the  twelve  parasyphilitics  showing  cardiovas- 
cular lesions,  eleven  gave  positive  luetin  reactions  and 
one  reacted  negatively.  Only  seven  showed  positive 
Wassermann  reactions.  The  one  case  giving  a  nega- 
tive luetin  test  had  a  positive  Wassermann  reaction. 
But  six  of  this  series  giving  negative  Wassermann 
reactions  gave  positive  luetin  tests.  It  is  in  this  group 
of  cases  in  which  great  care  must  be  exercised,  as  one 
might  readily  consider  the  test  negative  if  it  were  not 
watched  for  some  time.     Six  of  these  twelve  cases 


SERUM  DIAGNOSIS  OF  SYPHILIS.  205 

showed  reactions  of  the  torpid  form,  no  sign  of  the 
reaction  appearing  till  the  ninth  day,  and  in  one  case 
of  abdominal  aneurism,  which  gave  a  positive  Wasser- 
mann  test,  there  was  no  manifestation  until  the 
twenty-eighth  day.  The  luetin  reaction  will  prove  of 
great  value  in  this  type  of  case,  if  properly  followed, 
because  the  Wassermann  tests  are  relatively  incon- 
stant. "  Umstimmung  "  is  infrequent  here  as  com- 
pared with  cases  of  parasyphilis  of  the  central  ner- 
vous system.  No  other  cases  showing  constitutional 
symptoms  were  noted. 

Latent  Syphilis. — This  stage  of  syphilis  shows  a 
wonderful  constancy  of  results  with  this  test.  In  the 
23  cases  of  latent  syphilis  tested  all  gave  positive  re- 
actions. The  Wassermann  reaction  in  this  series  was 
positive  only  in  ten  cases.  In  three  of  these  cases  the 
Wassermann  reaction  on  the  cerebrospinal  fluid  was 
also  negative. 

Some  of  the  cases  reported  in  this  group  denied 
having  contracted  syphilis,  but  after  the  luetin  test 
was  found  positive,  in  several  instances  the  patients 
confessed  having  had  a  primary  lesion  or  secondary 
manifestations.  The  test  was  applied  in  four  cases  of 
pregnancy,  three  of  which  were  completed  by  the 
birth  of  macerated  foetuses  and  the  fourth  by  the 
birth  at  term  of  a  still-born  infant.  All  four  patients 
denied  infection.  The  three  former  patients  reacted 
"  violently "  to  the  luetin  and  showed  "  Umstim- 


206  SERUM  DIAGNOSIS  OF  SYPHILIS. 

niung."  The  Wassermann  reaction  was  positive  and 
an  anatomical  diagnosis  of  syphilis  in  the  infant  was 
made  in  each  case.  The  fourth  case  gave  a  negative 
luetin  reaction,  the  Wassermann  reaction  was  nega- 
tive, and  no  signs  of  syphilis  could  be  discovered  in 
the  dead  infant.  No  constitutional  disturbances  were 
noted  in  these  cases. 

Of  the  remaining  nineteen  cases  in  this  series, 
nine  showed  "  Umstimmung."  One  patient  suffer- 
ing from  carcinoma  of  the  head  of  the  pancreas,  who 
had  a  chancre  in  1865,  showed  a  delayed  luetin  test, 
but  gave  a  negative  Wassermann  reaction. 

Constitutional  Symptoms. — Constitutional  symp- 
toms consisting  of  tender  axillary  glands,  tender 
arms,  tachycardia,  abdominal  pains,  bone  pains,  and 
nausea  were  present  in  four  of  the  cases,  but  these 
symptoms  were  usually  of  only  24  to  48  hours'  dura- 
tion and  caused  no  alarming  discomfort  to  the  patient. 
One  case  showed  a  second  hemorrhagic  pustule  two 
weeks  after  the  first  manifestation  had  subsided. 

Thus  latent  syphilis,  so  difficult  to  diagnose  clin- 
ically and  in  which  the  Wassermann  reaction  is  not 
of  assistance  in  over  40  to  70  per  cent,  of  the  cases, 
shows  a  positive  luetin  reaction  in  all  the  cases  tried. 
It  seems  that  the  luetin  reaction  will  prove  of  even 
greater  value  in  the  diagnosis  of  this  group  than  in 
the  tertiary  stage  in  which  the  Wassermann  reaction 
is  more  constantly  present.    The  effects  of  treatment 


SERUM  DIAGNOSIS  OF  SYPHILIS.  207 

on  the  results  of  the  luetin  test  in  latent  syphilis  have 
not  been  significant. 

"  Umstimmung ." — In  reviewing  the  whole  series 
of  one  hundred  and  fifty  cases,  another  interesting 
fact  was  noted — that  in  many  cases  of  tertiary  and 
of  latent  syphilis  the  site  of  the  control  injection 
showed  almost  as  marked  a  reaction  as  developed 
about  the  point  where  the  luetin  was  injected.  This, 
as  was  pointed  out  by  Neisser  and  Bruck,  and  which 
seems  to  be  confirmed  by  Noguchi  and  this  series 
of  tests,  appears  to  be  due  to  the  susceptibility  to 
trauma  of  the  skin  of  syphilitics  late  in  the  disease 
("  Umstimmung  "),  for  not  one  of  the  seventy  con- 
trol patients  reacted  on  the  side  in  which  the  control 
emulsion  was  injected. 

The  cases  exhibiting  the  most  marked  "  control  " 
reactions  ("Umstimmung")  were  those  suffering 
from  syphilis  in  its  later  stages.  The  inconstancy  of 
this  phenomenon  in  all  probability  precludes  its  being 
of  an  allergic  nature,  which  latter  Noguchi  has  so 
well  shown  the  principle  of  the  luetin  reaction  to  be. 

Wolfsohn  drew  the  following  conclusions; 

1.  The  luetin  reaction  is  specific  for  syphilis. 

2.  The  reaction  is  found  of  greatest  value  in  the 
latent  and  tertiary  stages  of  the  disease. 

3.  In  some  treated  cases  of  secondary  syphilis  the 
reaction  is  positive. 

4.  In    parasyphilitics    with    the    cardiovascular 


208  SERUM  DIAGNOSIS  OF  SYPHILIS. 

manifestations  of  the  disease  the  reaction  may  be  de- 
layed for  from  nine  to  thirty  days. 

5.  The  luetin  reaction  is  helpful  in  the  diagnosis  of 
latent  syphilis  in  pregnancy. 

6.  The  state  of  "  Umstimmung  "  is  well  brought 
out  in  the  tertiary  and  latent  forms  of  syphilis. 

In  pediatry  the  luetin  reaction  seems  to  find  its 
useful  application.  Thus  Alan  Brown,  working 
under  Professor  L.  Emmet  Holt,  at  the  Babies'  Hos- 
pital, New  York,  obtained  a  positive  reaction  in  30 
(90  per  cent.)  out  of  the  33  children  suffering  from 
congenital  syphilis,  while  the  remaining  three  cases 
gave  doubtful  reactions.  In  73  non-syphilitic  chil- 
dren there  was  no  positive  reaction.  In  15  positive 
cases  the  reaction  was  of  pustular  form  and  the  other 
15  showed  the  inflammatory  nodules.  When  ar- 
ranged according  to  the  age  the  reactions  were  found 
as  follows: 

Positive  reaction  Doubtful  reaction 

1  to  3  months    12  3 

3  to  6  months    8 

6  to  12  months    6 

1  to  4  years  4 

30  3 

It  was  also  found  that  the  more  energetic  the 
treatment  the  more  distinct  was  the  luetin  reaction. 
The  three  infants  who  gave  the  indecisive  reactions 
had  not  been  under  the  antisyphilitic  treatment,  while 


SERUM  DIAGNOSIS  OF  SYPHILIS. 


209 


the  other  30  positive  cases  were  being  treated  either 
with  mercury  or  salvarsan  or  both  in  combination  for 
some  time. 

The  varieties  of  control  cases  studied  by  Brown 
are  shown  in  the  following  list.  The  Wassermann'^  as 
well  as  luetin  reaction  was  uniformly  negative  in 
these  cases. 

Table  30. 
NON-SYPHILITIC  CASES  FROM  THE  BABIES'  HOSPITAL. 
No.  cases  Disease 

1 Scleroderma 

2 Spastic  diplegia 

4 Marasmus 

2 Cretinism 

13 Bronchopneumonia 

2 Mongolian  idiocy 

2 Eczema 

1 Bronchitis 

1 Retropharyngeal  abscess 

7 Lobar  pneumonia 

16 Infant  feeding 

3 Tetanus 

3 Gastro-enteritis 

1 Pemphigus   neonatorum 

1 Scarlet  fever 

3 Empyema 

1 Tuberculous  meningitis 

1 Adenitis 

1 Still's  disease 

2 Rachitis 

1 Naevus 

1 Cleft  palate 

2 Hernia 

2 Congenital  heart 

73 

^  The  Wassermann  tests   (Noguchi  system)   were  performed  by  Dr. 
Bronfenbrenner  at  the  Rockefeller  Institute.    The  tests  were  made  abso- 
lutely objectively. 
15 


210  SERUM  DIAGNOSIS  OF  SYPHILIS. 

The  results  of  study  by  Brown  thus  establish  the 
specificity  of  the  luetin  reaction  for  syphilis  in  pedi- 
atric cases. 

THE    CONSEQUENCE    OF   THE   REPEATED   LUETIN   TESTS, 

When  a  patient  who  gives  a  positive  luetin  reac- 
tion is  tested  again  after  an  interval  of  time  not 
shorter  than  one  month  from  the  previous  test,  the 
reaction  appears  very  much  in  the  same  manner  as  the 
previous  one,  but  if  the  tests  are  repeated  a  number 
of  times  at  shorter  intervals,  the  reactions  seem  to 
appear  somewhat  quicker  than  the  earlier  ones. 
Apparently  there  is  an  abbreviation  of  the  incubation 
period  after  repeated  inoculations  of  the  luetin.  The 
duration  of  the  reaction  also  becomes  shorter.  A  nor- 
mal rate  of  reaction  velocity  seems  to  re-establish  once 
more,  if  the  patient  is  allowed  to  go  without  the 
test  for  a  few  months.  It  has  also  been  found  by 
Cohen  that  when  the  injection  of  the  luetin  is  made 
within  one  week  after  a  positive  reaction  was  ob- 
tained the  second  inoculation  may  produce  only  a 
transient,  mild  reaction  within  24  hours  and  quickly 
fades  away.  This  is  probably  due  to  the  negative 
phase  of  allergy  following  the  positive  reaction. 
(Plate  VI.) 


Plate  VI. 


Female,  aged  15  years.  A  typical  case  of  tardiac  congenital  sypliilis  with  bilateral 
insterstitial  keratitis,  Hutchinson  teeth,  saddle  nose,  etc.  Has  been  under  mercurial 
treatment.  This  case  has  shown  a  most  pronounced  pustular  reaction  when  tested  on 
several  previous  occasions,  but  the  present  test,  which  was  made  within  one  week  since 
the  last,  shows  only  a  mild  reaction. 


SERUM  DIAGNOSIS  OF  SYPHILIS.  211 

EFFECT  OF  TREATMENT  UPON  THE  LUETIN  REACTION. 

From  what  has  been  presented  in  the  foregoing 
it  is  quite  evident  that  the  luetin  reaction  remains  but 
little  affected  by  the  usual  intermittent  mercurial 
treatment  in  a  large  number  of  cases.  As  aforesaid 
the  allergic  condition  may  be  brought  out  by  the  usual 
mercurial  or  any  other  anti-syphilitic  treatment  in 
those  cases  where  it  has  not  appeared  yet.  Accord- 
ing to  my  observations,  salvarsan,  either  singly  or 
combined  with  mercury,  appears  to  exert  the  great- 
est influence,  not  only  in  inducing  the  development 
of  the  allergy,  but  also  in  removing  it  after  it 
has  developed.  The  explanation  for  this  remarkable 
feature  of  salvarsan  lies,  no  doubt,  in  its  powerful 
spiroch^etocidal  property,  by  virtue  of  which  it  de- 
stroys a  large  number  of  the  pallidum,  thus  induc- 
ing the  allergic  condition  in  earlier  stages  of  syphilis 
and  then,  if  successful,  annihilating  all  pallida,  thus 
removing  the  source  of  this  condition.  The  latter  is 
a  cure,  in  other  words. 

Although  there  can  be  no  dispute  about  an  ac- 
complished cure  with  the  mercurial  treatment  in  cer- 
tain percentages  of  cases  under  favorable  condi- 
tions, yet  the  successes  are  rather  uncertain.  Still  too 
early  for  deciding  a  cure  or  a  mere  latency  in  the 
cases  treated  with  salvarsan,  yet  I  am  strongly  in- 


212  SERUM  DIAGNOSIS  OF  SYPHILIS. 

clined  to  believe  that  there  are  certain  cases  which  are 
definitely  cured.  The  absence  of  the  clinical  and  sero- 
logical signs  of  syphilis  over  a  period  of  one  year  is 
certainly  an  encouraging  aspect,  but,  considering  the 
possibility  of  these  signs  being  absent  in  some  latent 
cases,  one  has  a  right  to  hesitate  in  pronouncing  these 
cases  as  cured.  It  is  in  this  connection  that  the  luetin 
reaction  may  become  a  great  aid  in  settling  this 
important  question.  As  already  stated,  the  luetin 
reaction  alone  cannot  decide  the  point,  but  combined 
with  other  means  of  diagnosis  it  is  bound  to  throw 
some  light  into  this  problem. 

I  have  now  certain  data  which  may  aid  to  clear  up 
the  relation  between  the  luetin  reaction,  clinical 
symptoms  and  Wassermann  reaction,  in  regard  to 
prognosis  of  syphilis.  In  one  series  of.  cases  the 
patients  were  treated  with  both  mercury  and  salvar- 
san,  and  in  due  time  the  clinical  and  the  serological 
symptoms  had  cleared  up  and  remained  so  for  a  cer- 
tain length  of  time.  The  luetin  reaction  was  tried  on 
each  of  them  and  the  results  are  as  follows : 

CASES   PARTIALLY  TREATED  WHERE    THE    CLINICAL  SYMPTOMS   ARE    ABSENT 
AND   THE    WASSERMANN    MOSTLY    NEGATIVE. 

Cases  of  Captain  Schmitter. 
1. — A.  G.,  male,  aged  33.  Chancre,  1906.  Keratitis,  1907,  and  re- 
currence in  1910  and  1911.  Treatment:  Injected  0.3  gm.  sal- 
varsan,  Feb.  14,  1911;  intravenously.  Mar.  18,  1911;  intramusc.  Mar. 
18,  1911;  intravenously,  July  29,  1911.  Wassermann:  Positive,  Nov. 
12,     1910;     negative     (?)     Feb.    23,     1911,     and     April    21,     1911; 


SERUM  DIAGNOSIS  OF  SYPHILIS.  213 

positive,  April  96,  1911;  negative.  May  2,  1911,  May  10,  1911, 
May  17,  1911,  June  1,  1911;  positive,  June  14,  1911;  negative  (?), 
July  13,  Juiy  20,  1911;  negative,  July  27,  1911.  Hg  and  KI 
treatment  from  Oct.,  1910,  to  Feb.,  1911.  Luetin  positive,  Sept. 
12,   1911. 

2. — W.  B.  J.,  male,  aged  24.  Chancre,  Dec,  1910.  Mucous  patches 
on  tonsils  and  soft  palate.  Mar.  16,  1911.  Treatment:  Injected 
salvarsan  intravenously  Mar.  16,  1911 ;  intramuscularly,  Mar.  16, 
1911.  Wassermann:  Positive,  Mar.  20,  1911,  and  April  12, 
1911;  negative.  May  22,  1911,  and  Aug.  29,  1911.  Luetin  positive, 
Sept.  3,  1911. 

3. — A.  B.  F.,  male,  aged  23.  Non-specific  (control).  Luetin  negative 
Sept.  1,  1911. 

4. — D.  J.  C,  male,  aged  33.  Chancre,  Nov.,  1910.  Secondaries  Jan.  16, 
1911.  Treatment:  Injected  salvarsan  intramusc.  and  intraven- 
ously, Feb  27,  1911;  intravenously,  Aug.  18,  1911.  Hg.  and  KI 
treatment  to  present  time.  Wassermann:  Positive  Feb.  1,  1911; 
±  Feb.  23,  1911;  negative.  Mar.  11,  1911,  and  Mar.  22, 
1911;  negative  June  19,  1911.  Luetin  positive  Sept.  6, 
1911. 

5. — E.  C,  male,  aged  33.  Chancre,  Apr.,  1904.  Secondaries  Aug., 
1904.  Hg.  for  3  months.  Treatment:  Injected  salvarsan  intra- 
muscularly and  intravenously,  Feb.  2,  1911.  Wassermann:  Positive, 
April  13,  1910,  and  Nov.  30,  1910;  negative  (?),  Feb.  23,  1911,  and 
Mar.  15,  1911;  negative.  Mar.  22,  1911,  and  June  28,  1911.  Luetin 
positive,  Sept.  8,  1911. 
6. — O.  C,  male,  aged  28.  Chancre,  Mar.,  1911.  Secondaries,  June  5, 
1911.  Treatment:  Injected  salvarsan  intravenously,  June  5,  1911; 
intramusc,  June  8,  1911.  Wassermann:  Positive  (?),  May  17, 
1911;  negative.  May  22,  1911;  positive,  June  1,  1911,  and  June  16, 
1911;  negative,  June  21,  1911,  and  June  28,  1911;  positive,  July  6, 
1911;  negative,  July  20,  1911,  and  July  27,  1911.  Luetin  doubtful, 
Sept.  5,  1911. 
7. — G.  C.  C,  male,  aged  23.  Chancre,  Jan  14,  1911.  Secondaries 
Feb.  20,  1911.  Treatment:  Injected  salvarsan  intramusc.  and  in- 
travenously, Feb.  24,  1911,  Hg.  and  KI  from  July  12,  1911,  to  the 
present.  Salvarsan  intravenously  Aug.  13,  1911.  Wassermann:  Nega- 
tive, Feb.  1,  1911;  negative  (?),  Feb.  17  and  Mar.  11,  1911;  negative. 
Mar.  22,  1911,  Mar.  29,  1911,  April  5,  1911,  April  12,  1911;  doubt- 
ful June  1  and  7,  1911;  positive,  July  6,  1911.  Luetin  positive  Sept. 
3,  1911. 

8. — S.  F.,  male,  aged  28.  Chancre,  June,  1910.  Secondaries,  Oct.  28, 
1910.     Treatment:     Injected   salvarsan   intramuscularly   and   intra- 


214  SERUM  DIAGNOSIS  OF  SYPHILIS. 

venousiy,  Feb.  13,  1911.  Wassermann:  Negative  Oct.  28,  1910; 
positive  Feb.  13,  1911;  negative,  Feb.  23,  1911,  Mar.  22,  1911,  and  July 
15,  1911.     Luetin  positive,  Sept.  11,  1911. 

9. — T.  A.  K.,  male,  aged  25.  Chancre,  Jan.,  1911.  Secondaries,  Feb. 
26,  1911.  Treatment:  Injected  salvarsan  intramusc.  and  intra- 
venously, Feb.  26,  1911;  intravenously,  Aug.  19,  1911.  Hg.  and 
KI  from  July  28,  1911,  to  present.     Luetin  positive,  Sept.  5,  1911. 

10. — J.  H.  McB.,  male,  aged  29.  Chancre,  Feb.  1,  1911.  Secondaries 
April  13,  1911.  Treatment:  Injected  salvarsan  intravenously, 
April  11,  1911;  intramuscularly,  April  13,  1911.  Wassermann: 
Positive,  April  13,  1911,  April  21,  1911,  and  April  26, 
1911;  negative,  May  17,  1911,  and  June  7,  1911.  Luetin  positive, 
Sept.    7,    1911. 

12. — J.  J.  M.,  male,  aged  28.  Chancre,  Apr.,  1911.  Secondaries,  Aug. 
17,  1911.  Treatment:  Injected  salvarsan  intravenously,  Aug.  18, 
1911.  Wassermann:  Negative  Aug.  23  and  Aug.  26,  1911.  Luetin 
positive,  Sept  6,  1911. 

13. — J.  G.  M.,  male,  aged  30.  Macular  rash  over  body  in  1900.  Treat- 
ment; Injected  salvarsan  intravenously  May  17,  1911;  intramusc. 
May  10,  1911.  Wassermann:  Positive  May  2,  1911;  negative  (?) 
May  17,  1911;  negative  May  22  and  June  1,  1911;  positive  June 
7,  1911,  and  June  14,  1911;  negative  June  21,  1911.  Luetin  posi- 
tive,   Sept.    3,    1911. 

14._W.  M.,  male,  aged  28.  Chancre,  1910.  Secondaries,  April  10,  1911. 
Treatment:  Injected  salvarsan  intravenously,  April  10,  1911; 
intramusc.  Apr.  13,  1911;  intravenously,  Aug.  20,  1911.  Hg  and 
KI  to  the  present  time.  Wassermann:  Negative,  Mar.  27,  1911; 
positive  (?),  April  3,  1911;  positive,  April  12,  1911;  negative, 
April  21,  1911;  negative  (?),  April  26,  1911;  positive.  May 
2,  1911;  O  May  10,  1911;  positive  (?),  May  17,  1911;  negative, 
June  1,  1911;  positive,  June  7,  1911;  positive,  June  14,  1911.  Luetin 
positive,  Sept.  7,  1911. 

15. — F.  S.,  male,  aged  25.  Chancre,  Dec,  1910.  Secondaries,  Feb., 
1911.  Treatment:  Injected  salvarsan  intramusc.  and  intravenously, 
Feb.  13,  1911;  intramusc,  May  20,  1911.  Hg  and  KI  from  May 
27  to  Aug.  1,  1911.  Wassermann:  Positive,  Feb.  10  and  Feb.  23, 
1911;  positive  (?),  Mar.  11,  1911;  negative.  Mar.  22,  1911;  posi- 
tive. May  12,  1911;  negative,  Aug.  23,  1911.  Luetin  positive,  Sept. 
6,    1911. 

16.— R.  W.,  male,  aged  26.  Chancre,  Oct.,  1909.  Secondaries,  Aug. 
21,  1911.  Treatment:  Injected  salvarsan  intravenously,  Aug.  21, 
1911.  Hg  and  KI  from  Aug.  23,  1911,  to  date.  Wassermann: 
Negative   Aug.   26,    1911.      Luetin  positive   Sept.   6,    1911. 


SERUM  DIAGNOSIS  OF  SYPHILIS.  215 

17.— J.  H.  S.,  male,  aged  29.  Chancre,  Jan.,  1909.  Secondaries,  July 
22,  1911.  Treatment  not  given.  Wassermann:  Negative  July  27, 
1911;  positive  (?),  Aug.  5,  1911;  negative,  Aug.  23  and  Aug.  29, 
1911.      Luetin   negative,    Sept.    6,    1911. 

18. — H.  L.  A.,  male,  aged  22.  Congenital.  Treatment:  Injected 
salvarsan  intravenously,  June  5,  1911;  intramusc,  June  8,  1911. 
Wassermann:  Positive,  June  1  and  June  21,  1911;  negative,  June 
29,  1911;  positive,  July  6,  1911;  negative,  July  13,  July  20  and 
July   27,    1911.      Luetin   positive,   Sept.    11,  1911. 

19.— P.  F.  B.,  male,  aged  26.  Chancre,  Feb.  19,  1910.  Secondaries, 
May  16,  1911.  Treatment:  Injected  salvarsan  intramusc.  and 
intravenously,  Feb.  17,  1911;  intravenously  Aug.  18,  1911.  Hg 
and  KI  from  July  20,  1911,  to  present  time.  Wassermann:  Positive, 
Feb.  17,  Feb.  23,  Mar.  11,  1911;  negative.  Mar.  22  and  July  6, 
1911.     Luetin  doubtful,  Sept.  5,  1911. 

20. — G.  A.  D.,  male,  aged  30.  Chancre,  May  12,  1911.  Secondaries, 
July  1,  1911.  Treatment:  Injected  salvarsan  intravenously,  July  1, 
1911;  intramusc,  July  4,  1911.  Hg  and  KI  from  July  27, 
1911,  to  the  present.  Wassermann:  doubtful,  July  6,  1911;  positive 
(?),  July  13,  1911;  positive  (?),  July  20,  1911;  doubtful,  July  27, 
1911.     Luetin    doubtful,    Sept.    6,    1911. 

21. — C.  N.  S.,  male,  aged  26.  Chancre,  Mar.  10,  1911.  Secondaries, 
Apr.  2,  1911.  Treatment:  Injected  salvarsan  intravenously,  Aug. 
19,  1911;  intravenously  and  intramuscularly,  April  6,  1911.  Hg 
and  KI  from  July  27,  1911,  to  present.  Wassermann:  Positive,  April 
10,  1911;  positive  April  21,  1911;  positive,  April  26,  1911;  posi- 
tive. May  2,  1911;  doubtful.  May  10,  1911;  negative.  May  17,  May  22, 
and  June  1,  1911;  doubtful,  July  20,  1911;  negative,  July  27,  1911. 
Luetin  negative,  Sept.  6,  1911. 

T'he  above  series  of  cases  illustrate  very  clearly 
that  the  absence  of  the  clinical  and  serological  signs 
of  syphilis  indicates  merely  that  they  were  mostly  in 
the  latency  of  the  disease  and  could  be  detected  by  the 
positive  luetin  reactions. 

In  another  series  of  cases  (Dr.  Corbus)  the 
patients  were  treated  similarly,  but  decidedly  more 
vigorously  and  effectively.     The  patients  remained 


216  SERUM  DIAGNOSIS  OF  SYPHILIS. 

symptomless  and  their  Wassermann  reactions  nega- 
tive for  varying  lengths  of  time  when  the  luetin  re- 
action was  applied. 


CASES     THOROUGHLY     TREATED     WHERE     THE     CLINICAL     SYMPTOMS 
AND   THE    WASSERMANN    ARE   ABSENT. 

Private  cases  of  Dr.  Corbus. 

1. — C.  K.  A.,  male,  aged  43,  early  secondaries  when  he  came  under 
observation.  Now  believed  cured.  Treatment:  Injected  intramusc. 
0.5  gm.  salvarsan  Nov.  4,  1910,  and  Jan.  17,  1911;  intravenously,  July 
24,  1911.  Vigorous  Hg  rubbings  between  injections.  Wassermann 
negative,  Jan.  17,  1911,  March  6,  1911,  June  8,  1911,  July  25, 
1911,  and  Dec.  1,  1911.  Luetin  negative.  Mar.  14,  1912.  Com- 
plained slight  gastric  disturbance. 

2. — ^W.  E.  M.,  male,  aged  28,  latent  lues.  Treatment:  Salvarsan  in- 
jected abroad;  intramusc,  June  21,  1911,  Aug.  15,  1911;  intra- 
venously, Oct.  27,  1911,  Dec.  18,  1911.  Wassermann:  Positive, 
June  9,  1911;  negative,  Sept.  12,  1911;  negative  (?),  Dec.  19, 
1911;  negative.  Mar.  8,  1912.    Luetin  negative.  Mar.  14,  1912. 

3. — M.  H.,  male,  aged  22,  latent.  Treatment:  Injected  salvarsan 
intravenously,  July  24,  1911,  and  Sept.  8,  1911.  Wassermann 
negative,    Sept.    12,    1911.      Luetin    negative.    Mar.    14,    1912. 

4.— G.  C.  F.,  naale,  aged  27,  early  secondaries  when  he  came  under  ob- 
servation. No  symptoms  now.  Treatment:  Injected  salvarsan 
intramuscularly  Mar,  9,  1911,  and  April  20,  1911;  intravenously 
Oct.  27,  1911.  Vigorous  Hg  rubbings  between  injections.  Wasser- 
mann negative,  Sept.  8,  1911.  Luetin  positive,  Mar.  14,  1912. 
5. — B.  B.,  female,  aged  20,  early  secondaries  when  she  came  under 
observation.  No  symptoms  now.  Treatment:  Injected  salvarsan 
intramusc,  Oct.  18,  1910,  and  Nov,  30,  1911.  Wassermann:  Posi- 
tive, Oct.  18,  1910,  and  Nov,  11,  1910;  negative,  Dec  1,  1910; 
slightly  positive.  Mar.  8,  1912.     Luetin  positive.  Mar.   14,  1912. 

6. — C.  E.  H.,  male,  aged  26,  latent.  Treatment:  Injected  salvarsan 
intramusc,  Dec  9,  1910;  intravenously,  April  20,  1911.  Wasser- 
mann: Negative,  Mar.  16,  1911,  April  20,  1911,  June  14,  1911, 
Dec.  1,  1911.  Luetin  negative,  Mar.  14,  1912. 
7. — M.  E.,  male,  aged  34.  Now  believed  cured.  Treatment:  Injected 
salvarsan  intravenously  Nov.  18,  1910;  intramusc.  Nov  21,  1910; 
intravenously  Aug.  11,  1911,  Nov.  24,  1911,  and  Mar.  1,  1912. 
Wassermann:     Positive,    Nov.    11,    1910,    Nov.    28,    1910,    Dec    15, 


SERUM  DIAGNOSIS  OF  SYPHILIS.  217 

1910,  and  Mar.  8,  1911;  negative,  Sept.  18,  1911,  Oct.  2T,  1911, 
Nov.  28,  1911,  and  Mar.  2,  1913.  Luetin  negative,  Mar.  14, 
1912, 

8. — W.  S.,  male,  aged  29,  latent.  Treatment:  Injected  salvarsan 
intramusc.  Jan.  12,  1911;  intravenously.  Mar.  22,  1911,  and  Aug. 
21,  1911.  Vigorous  Hg  rubbings  between  injections.  Wassemiann: 
Negative  Mar,  2,  1911;  slightly  positive  Aug.  1,  1911;  negative 
Aug,  9,  1911,  and  Oct.  24,  1911.'  Luetin  negative.  Mar.  14, 
1912. 

9. — A,  E,  B.,  male,  aged  34,  Apparent  mercury  cure.  Treatment 
with  vigorous  Hg  rubbings  and  injections  regularly,  Wassermann: 
Positive  May  1,  1911;  negative  May  11,  1911  (Dr,  Waugh) ; 
negative  May  13,  1911,  June  10,  1911,  and  Jan.  1,  1912.  Luetin 
negative  Mar,  14,  1912, 
10, — F,  R.,  male,  aged  33,  late  secondaries  when  he  came  under  ob- 
servation. Latent  at  present.  Treatment:  Injected  salvarsan 
intramusc.  Nov.  17,  1910,  and  Jan.  13,  1911;  intravenously,  Dec.  1, 

1911.  Vigorous  Hg  rubbings  between  injections.  "Wassermann: 
Positive  Jan.  11,  1911,  and  Mar,  18,  1911;  negative  Sept.  27, 
1911,  and   Nov.   28,   1911,     Luetin  positive,  Mar,   14,   1912. 

11, — E,  H,  J,,  male,  aged  28,  luetic  periostitis  when  he  came  under 
observation.  Latent  at  present.  Treatment:  Injected  salvarsan 
intravenously  May  14,  1911;  intramusc.  May  20,  1911;  intravenously 
Nov.  11,  1911.  Vigorous  Hg  rubbings  between  injections  and  KI, 
Wassermann:  positive,  April  24,  1911,  negative,  Oct,  21,  1911,  and 
Feb,  23,  1912.     Luetin  negative.  Mar.  14,  1912. 

12. — L.  A,  R.,  male,  aged  36,  believed  cured.  Treatment:  Hg  rubbings 
and  injections,  chronic  intermittent  form.  Wassermann:  Nega- 
tive, Mar.  20,  1912.     Luetin  negative.  Mar,  14,  1912, 

13. — G.  S.,  male,  aged  20,  primary  lesion  when  he  first  came  under 
observation,  Wassermann  always  negative.  Treatment:  Injected 
salvarsan  intravenously  April  29,  1911;  intramusc.  May  4,  1911; 
intravenously  July  1,  1911,  and  July  24,  1911,  Hg  rubbings  for 
two   months.      Wassermann   negative   June    10,    1911,   and   Oct.   31, 

1911.  Luetin    negative.    Mar.    14,    1912. 

14. — J,  c.  S.,  male,  aged  40,  Believed  cured  after  biological  method. 
Treatment:  Injected  salvarsan  intramusc.  Nov.  24,  1910,  and  ? 
date;  intravenously  May  7,  1912.  Wassermann:  Negative  Nov.  11, 
1910,  May  9,   1911,  Sept,  27,   1911,    (?)    Nov,   7,1911,  and  April  9, 

1912,  Luetin,  indolent  reaction,  marked  after  21  days.  Mar,  14, 
1912.      Wassermann   negative   at   this   time, 

15. — H,  C.  S.,  male,  aged  25,  latent  syphilis.  Treatment:  Injected 
salvarsan    intramusc.    6    gm.,    May    19,    1911,    and    Aug.    28,    1911; 


218  SERUM  DIAGNOSIS  OF  SYPHILIS. 

intravenously  Nov.  10,  1911,  and  Mar.  24,  1912.  Hg  rubbings' 
between  injections.  Wassermann:  Positive  May  8,  1911;  slightly 
positive,  Aug.  30,  1911;  slightly  positive,  Oct.  20,  1911;  negative, 
Feb.   6,   1912.     Luetin  negative.  Mar.   14,  1913. 

16. — L.  S.  K.,  male,  aged  ?,  secondary  lues  when  he  came  under  ob- 
servation. Latent  at  present.  Treatment:  Injected  salvarsan 
Sept.  11,  1911;  intravenously,  Oct.  30,  1911,  Dec.  23,  1911,  and 
Mar.  4,  1912.  Hg  rubbings  between  injections.  Wassermann: 
Positive,  Sept.  12,  1911,  and  Oct.  31,  1911;  negative,  Dec.  24, 
1911.      Luetin    negative.    Mar.    14,    1912. 

17. — F.  McN,  male,  aged  32,  now  believed  cured.  Treatment:  In- 
jected intramusc.  salvarsan,  Nov.  17,  1910,  and  Jan.  12,  1911; 
intravenously  Oct.  30,  1911.  Wassermann:  Positive,  Jan.  8,  1911, 
negative  (?),  April  22,  1911,  negative  (?),  Sept.  27,  1911,  negative 
(?),   Oct.   31,   1911.     Luetin   negative.   Mar.   14,   1912. 

18. — W.  D.,  male,  aged  21,  secondaries  when  he  came  under  observation. 
Latent  now.  Treatment:  Injected  salvarsan  intramusc.  Mar.  13, 
1911,  and  May  8,  1911;  intravenously  July  7,  1911.  Hg  rubbings 
between  injections.  Wassermann  positive,  July  7,  1911,  Luetin 
positive.  Mar.   14,  1912. 

19. — J.  K.,  male,  aged  28,  latent  now.  Treatment:  Injected  salvarsan 
intramusc.  Feb.  16,  1911;  intravenously  July  24,  1911,  and  Nov.  3, 
1911.  Hg  rubbings  between  injections  and  Hg  internally.  Was- 
sermann:  Positive  Jan.  23,  1911,  and  May  1,  1911;  negative  Aug.  25, 

1911,  and  Nov.  7,  1911.    Luetin  negative.  Mar.  14,  1912. 

20. — ^W.  H.  S.,  male,  aged  22.  Early  secondaries  when  he  came  under 
observation.  Latent  now.  Treatment:  Injected  salvarsan  in- 
tramusc, April  24,  1911,  and  Sept.  7,  1911;  intravenously  Jan.  19, 

1912.  Vigorous  mercury  rubbings  between  injections.  Wassermann: 
Negative  Sept.  8,  1911,  and  Jan.  23,  1912.  Luetin  negative.  Mar. 
14,   1912. 

The  above  series  of  cases,  though  necessarily 
limited  in  number,  are  very  important  from  the  prog- 
nostic standpoint  of  view,  as  they  are  exactly  the 
cases  where  the  clinicians  desire  to  ascertain  whether 
the  absence  of  the  clinical  and  serological  signs  of 
syphilis  is  an  indication  of  latency  or  a  cure.  That 
some  of  the  cases  of  this  group  of  patients  belong 


SERUM   DIAGNOSIS  OF  SYPHILIS.  219 

to  the  latent  stage  of  the  disease  is  well  shown  by  the 
positive  outcome  of  the  luetin  reaction,  yet  it  remains 
for  the  future  to  decide,  by  continued  observations 
for  years  to  come,  whether  or  not  the  other  cases 
where  the  luetin  reaction  as  well  as  the  clinical  and 
serological  symptoms  are  now  missing  had  really 
been  cured  by  the  energetic  treatment. 

While  reading  the  proof,  an  excellent  article  of 
Kammerer  has  appeared  in  the  Munchener  medi- 
^nische  Wochenschrift.  This  investigator,  working 
at  the  medical  clinic  of  Professor  von  Miiller,  at 
Munich,  Germany,  has  tried  out  the  luetin  submitted 
to  him  by  the  writer  on  a  number  of  cases  suffering 
from  various  diseases,  and  obtained  the  results,  which 
are  in  the  main  confirmatory  of  those  obtained  by 
other  investigators.  While  the  percentage  of  posi- 
tive reaction  in  syphilis  is  much  lower  than  that  found 
by  others,  Kammerer  found  the  reaction  to  be  spe- 
cific for  syphilis.  This  is  a  highly  important  factor, 
as  the  value  of  the  luetin  test  depends  upon  its 
specificity. 

Through  a  personal  communication  I  am  in- 
formed of  the  results  obtained  by  Goldenberg  and 
Kaliski,  who  applied  the  luetin  to  a  very  large  num- 
ber of  cases  at  the  Mount  Sinai  Hospital,  New  York. 
Their  observations  confirm  those  of  previous  workers. 


220  SERUM  DIAGNOSIS  OF  SYPHILIS. 

Fordyce,  Pusey,  and  others  have  also  obtained  simi- 
lar results  on  dermatological  cases. 

It  is  hoped  that  by  preparing  a  more  active  luetin 
it  may  eventually  be  possible  to  substitute  the  intra- 
demic  application  by  a  cutaneous  procedure,  in  order 
to  render  the  test  simple  enough  to  enable  more  phy- 
sicians to  employ  it.  At  present  the  mode  of  appli- 
cation of  the  luetin  test  is  by  no  means  ideal  and  re- 
quires much  care  and  preparation  to  perform  it 
properly. 


XV. 

THE  BUTYRIC  ACID  TEST. 

While  studying  the  relation  of  proteids,  lipoids 
and  salts  to  the  Wassermann  reaction,  I  observed  that 
the  syphilitic  antibody*  is  contained  in  or  precipitated 
with  globulin,  and  particularly  the  euglobulin  fraction 
of  the  blood-serum  or  cerebrospinal  fluid.  I  inci- 
dentally ascertained  that  the  globulin  fraction  of 
these  fluids  is  increased  in  syphihs,  and  that  there  ex- 
ists a  parallel  between  the  titre  of  the  syphilitic  anti- 
body and  the  amount  of  the  globulin  fractions,  to 
which  rule  I  observed  certain  exceptions,  in  which 
this  parallelism  was  absent.  There  would  therefore 
appear  to  be  no  necessary  connection  between  the 
syphilitic  antibody  and  the  increase  of  globulin, 
although  the  two  conditions  are  likely  to  be  associated. 
While  the  appearance  of  the  antibody  and  the  in- 
crease of  the  globulin  are  often  found  associated,  I 
have  observed  that  the  increase  in  the  globulin  is 
recognizable  earlier  than  the  presence  of  the  anti- 
body; and  in  the  early  stages  of  primary  syphihs, 
when  the  presence  of  the  antibody  may  not  be  de- 
tectable, the  globulin  content  is  seen  already  to  be 

*  The  term  antibody  in  this  instance  refers  to  the  active  lipotropic 
substances  causing  the  Wassermann  reaction,  but  not  the  specific 
antibody. 

221 


222  SERUM  DIAGNOSIS  OF  SYPHILIS. 

increased.  Again,  in  cases  of  latent  syphilis  the  anti- 
body so  far  as  it  is  demonstrable  is  more  or  less  incon- 
stant and  may  thus  escape  detection  altogether, 
whereas  it  is  exceptional  not  to  find  the  globulin  in- 
creased. These  facts  apply  also  to  the  cerebrospinal 
fluid.  In  specimens  of  cerebrospinal  fluid  coming 
from  cases  of  secondary  or  tertiary  syphilis  in  which 
there  is  no  special  involvement  of  the  central  nervous 
system,  the  syphilitic  antibody  is  extremely  difficult 
to  detect,  and  this  is  true  even  though  this  fluid  con- 
tains an  increase  in  its  protein  fraction.  In  cases  of 
parasyphilitic  affections,  where  the  central  nervous 
system  is  primarily  involved,  the  detection  of  the  anti- 
body is  often  readity  accomplished.  In  general  pa- 
ralysis, the  antibody  is  detectable  in  the  cerebrospinal 
fluid  in  about  90  per  cent,  of  the  cases.  In  locomotor 
ataxia  or  cerebral  or  spinal  syphilis,  the  detection  of 
the  antibody  in  the  cerebrospinal  fluid  is  successful 
in  about  60  per  cent,  of  the  cases.  The  increase  of 
protein  in  the  cerebrospinal  fluid  of  these  cases  is 
greater  than  the  appearance  of  the  antibody,  and  my 
experience  leads  me  to  conclude  that  the  abnormally 
high  protein  content  is  a  more  constant  occurrence 
in  the  cerebrospinal  fluid  in  syphilitic  and  parasyphi- 
litic cases  than  is  the  presence  of  a  detectable  antibody. 
For  the  detection  of  the  increase  of  globulin  in 
the  blood  or  cerebrospinal  fluid  numerous  methods 


SERUM  DIAGNOSIS  OF  SYPHILIS.  223 

have  been  devised  by  different  investigators.  For 
the  estimation  of  the  globuhn  of  the  blood,  the 
methods  proposed  are  the  usual  chemical  procedures, 
the  application  of  which  to  clinical  laboratories  has 
not  been  successfully  accomplished.  The  detection  of 
the  increased  globulin  in  the  cerebrospinal  fluid  has 
been  accomplished  by  simpler  quantitative  methods, 
such  as  the  half  saturation  with  ammonium  sulphate, 
as  devised  by  Nonne  and  Apelt,  which  is  applicable  to 
clinical  laboratories.  On  the  whole,  this  method,  while 
useful,  is  somewhat  difficult  of  application  and  fails 
to  give  a  proper  differentiation  when  the  increase  in 
the  globulin  is  only  slight.  My  own  method  consists 
in  the  employment  of  butyric  acid  as  precipitant  for 
the  globulin,  but  the  manner  of  application  differs 
somewhat  according  as  it  is  applied  to  the  blood-serum 
or  the  cerebrospinal  fluid.  This  method  of  detecting 
an  increase  in  the  globulin  content  is  applicable  es- 
pecially to  the  cerebrospinal  fluids  and  is  so  simple 
as  to  be  within  the  reach  of  the  simplest  laboratory. 

Method  for  Cerebrospinal  Fluid. — Two  parts 
of  the  cerebrospinal  fluid  to  be  examined  are 
mixed  with  5  parts  of  a  10  per  cent,  butyric  acid 
solution  in  physiological  salt  solution,  and  are  heated 
over  a  flame  and  boiled  for  a  brief  period.  One  part 
of  a  normal  solution  of  NaOH  is  then  added  quickly 
to  the  heated  mixture,  and  the  whole  boiled  once 


224  SERUM  DIAGNOSIS  OF  SYPHILIS. 

more  for  a  few  seconds.  The  actual  quantities  of 
these  three  agents  that  I  prefer  are  0.1  or  0.2 
c.c.  of  the  spinal  fluid,  0.5  c.c.  of  the  butyric  acid 
solution,  and  0.1  c.c.  of  the  normal  sodium  hydroxide. 
It  is  necessary  to  take  the  precaution  to  employ  for 
this  test  only  cerebrospinal  fluid  entirely  free  from 
blood. 

The  presence  of  an  increased  content  of  protein 
in  the  cerebrospinal  fluid  is  indicated  by  the  appear- 
ance of  a  granular  or  floccular  precipitate,  which 
gradually  settles  to  the  bottom  of  the  tube,  beneath 
a  clear,  supernatant  fluid.  The  velocity  and  intensity 
of  the  reaction  vary  according  to  the  quantity  of  the 
protein  contained  in  a  given  specimen.  The  greater 
the  amount  of  the  protein,  the  more  quickly  and  dis- 
tinctly the  reaction  appears.  The  granular  precipi- 
tate appears  within  a  few  minutes  in  a  specimen  con- 
taining a  considerable  increase  in  protein,  while  one 
hour  may  be  required  to  obtain  a  distinct  reaction 
in  specimens  weaker  in  protein.  In  obtaining  the 
reaction,  the  time  period  should  not  be  greater  than 
two  hours. 

This  reaction  I  have  found  to  appear  regularly 
in  the  cerebrospinal  fluid  of  the  patients  with  syphi- 
Htic  and  parasyphilitic  affections,  and  also  in  all  cases 
of  inflanmiation  of  the  meninges  caused  by  such 
micro-organisms  as  Diplococcus  intracellulariSj  pneu- 
mococcus,  influenza  bacillus,  tubercle  baciUus,   etc. 


SERUM  DIAGNOSIS  OF  SYPHILIS.  225 

These  acute  inflammatory  infections  are  of  course 
readily  differentiated  from  the  syphilitic  affections. 

According  to  the  recent  investigations  of  Flexner 
and  his  coworkers,  Lewis  and  Clark,  the  cerebrospinal 
fluid  of  man  as  well  as  monkeys  with  acute  poliomyeli- 
tis contains  an  abnormally  large  amount  of  proteins 
and  gives  a  positive  reaction  to  the  butyric  acid  test. 
It  was  found  that  the  increase  of  the  proteins  is  one 
of  the  earliest  symptoms  of  this  disease  and  reaches 
the  maximum  just  before  the  paralytic  symptoms 
develop.  Flexner  employed  the  test  in  determining 
the  result  of  inoculation  of  his  filtrable  virus  into 
monkeys.  He  also  discovered  a  certain  number  of 
abortive  cases  of  poliomyelitis  in  man  by  means  of 
the  butyric  acid  test  and  cytodiagnosis,  thereby  of 
course  eliminating  all  other  diseases  giving  the  same 
reaction  and  lymphocytosis  by  careful  clinical  data 
and  other  differential  diagnostic  means. 

Normal  cerebrospinal  fluid  gives  with  the  butyric 
acid  test  a  slight  opalescence  and  sometimes  a  marked 
turbidity,  but  the  granular  precipitate  does  not  occur 
at  all  or  occurs  only  after  several  hours  or  even  after 
twenty-four  hours. 

Method  for  Blood-serum. — One  part  of  clear 
serum  free  from  haemoglobin  is  mixed  with  9  parts 
of  a  half-saturated  solution  of  ammonium  sulphate, 
the  precipitated  globulin  centrifugahzed  by  a  power- 

16 


226  SERUM  DIAGNOSIS  OF  SYPHILIS. 

f ul  machine,  and  the  compact  globulin  fraction  sepa- 
rated by  decantation  from  the  supernatant  fluid.  The 
deposit  may  now  be  weighed  to  obtain  an  idea  of  its 
quantity.  It  is  then  redissolved  in  10  parts  of  0.9 
per  cent,  salt  solution,  and  is  ready  for  the  test. 
The  test  is  made  by  mixing  one  part  of  the  solution 
with  an  equal  part  of  10  per  cent,  butyric  acid  solu- 
tion, when  a  prompt,  dense,  milky  turbidity  appears 
in  the  mixture,  if  the  serum  tested  was  derived  from 
cases  of  syphilis,  while  it  remains  clear  or  shows  only 
a  slight  opalescence  without  precipitation  after 
several  hours'  standing,  if  it  was  derived  from  persons 
not  suffering  with  syphilis. 

In  carrying  out  this  test  I  have  been  in  the  custom 
of  using  0.5  c.c.  of  serum  and  4.5  c.c.  of  ammonium 
sulphate  solution,  and  of  performing  the  centrifu- 
galization  for  30  minutes  in  a  machine  which  runs 
at  the  rate  of  5000  revolutions  per  minute.  After 
decanting  the  fluid,  the  deposit  is  redissolved  in  5 
c.c.  of  0.9  per  cent,  salt  solution.  Of  this  solution, 
0.5  c.c.  is  mixed  with  an  equal  quantity  of  the  butyric 
acid  solution.  It  is  advisable  to  carry  out  the  ex- 
amination of  the  several  specimens  of  the  fluid  at  the 
same  time,  and  especially  to  include  a  normal  serum, 
giving  a  negative  reaction,  to  act  as  a  control  for  the 
series. 

It  should  be  mentioned  that  the  weights  of  the 


SERUM  DIAGNOSIS  OF  SYPHILIS.  227 

globulin  deposits  are  comparable  with  one  another 
only  when  they  are  packed  equally  by  a  definite  de- 
gree of  centrifugalization.  Under  similar  conditions 
the  use  of  weighing  to  determine  the  increase  in  the 
globulin  is  an  advantage  and  is  recommended,  but 
where  the  conditions  are  not  identical,  such  weighings 
may  give  an  erroneous  indication. 

BUTYRIC    ACID    REACTION    OF    CEREBROSPINAL    FLUID    IN 
SYPHILIS    AND    PARA8YPHILITIC    DISEASES. 

The  results  which  I  have  obtained  with  the 
butyric  acid  reaction,  along  with  Dr.  Moore  of 
the  Manhattan  State  Hospital  on  Ward's  Island, 
will  serve  to  illustrate  the  value  of  the  butyric 
acid  test  in  psychiatry.  For  the  purpose  of  the 
test  the  cerebrospinal  fluid  from  cases  of  general 
paralysis,  tabes  dorsalis,  dementia  precox,  epilepsy, 
alcoholic  psychosis,  senile  dementia,  and  certain 
other  forms  of  insanity  was  employed.  In  order 
to  arrive  at  an  accurate  result,  the  fluids  tested  by 
means  of  the  butyric  acid  were  also  subjected  to  the 
Wassermann  test  and  to  the  usual  cytodiagnosis  ob- 
servation. In  order  to  make  the  series  complete,  the 
cerebrospinal  fluid  from  several  cases  of  syphilis  free 
from  involvement  of  the  central  nervous  system  was 
also  examined. 

In  the  secondary  and  tertiary  stages  of  syphilis. 


SERUM  DIAGNOSIS  OF  SYPHILIS. 

without  direct  involvement  of  the  nervous  system,  the 
cerebrospinal  fluid  yielded  a  reaction  of  feeble  inten- 
sity to  the  butyric  acid  test.    These  fluids  gave  neither 

Table  31. — Results  in  cases  in  which  the  diagnosis  was  reasonably 

certain. 


No. 
of 

cases 

Butyric  acid 
reaction 

Wassermann 
reaction 

Cell  count 

+ 

- 

± 

1  + 

- 

± 

+ 

- 

± 

Syphilis: 
Secondary  stage. .  . 

(without  nervous 

symptoms) 
Tertiary  stage 

(without  nervous 

symptoms) 
Cerebral  syphilis.  . 
Spinal  syphilis.  . . . 
Hereditary  syphilis 
Parasyphilis : 
General  paralysis: 

Cerebral 

Tabetic 

Tabes 

3 

1 

3 

3 

10 

43 
17 
11 

3 

1 

3 
3 
9 

37 
17 
11 

0 

0 

0 
0 
0 

4 
0 
0 

0 

0 

0 
0 

1 

2 
0 
0 

0 

0 

1 
2 
8 

32 

12 

6 

3 

1 

1 

1 
2 

6 
3 

4 

0 

0 

1 
0 
0 

5 

2 
1 

0 

0 

3 
3 

39 
16 
11 

3 
1 

0 
0 

2 
1 
0 

0 

0 

0 
0 

2 
0 
0 

91 

84 

4 

3 

61 

21 

9 

Psychoses: 
Arteriosclerotic  . . . 

Traumatic 

Senile 

Epileptic 

Alcoholic 

3 

2 
1 
6 
7 
2 
11 
2 

1 
0 
0 
0 
0 
0 
1 
0 

2 
2 
1 
6 
6 
2 
10 
2 

0 
0 
0 
0 

1 

0 
0 
0 

1 
0 
0 
0 
3 
1 
1 
0 

2 
2 
1 
5 
3 
1 
8 
2 

0 
0 
0 
1 
1 
0 
2 
0 

1 
0 
0 
0 
0 
0 
1 
0 

2 
2 
1 
6 
6 
2 
10 
2 

0 
0 
0 
0 
1 

Manic  depressive. 
Dementia  prsecox  . 
Imbecility 

0 
0 
0 

34 

2 

31 

1 

6 

24 

4 

2 

31 

1 

a  positive  cytodiagnosis  nor  the  Wassermann  reaction. 
The  cerebrospinal  fluid  of  a  group  of  cases  of  heredi- 


SERUM  DIAGNOSIS  OF  SYPHILIS. 


229 


tary  syphilis  gave  a  positive  butyric  acid  reaction  in 
about  90  per  cent,  and  a  positive  Wassermann  re- 
action in  about  80  per  cent,  of  those  examined.  On 
the  other  hand  the  cerebrospinal  fluid  obtained  from 
cases  of  cerebral  and  spinal  syphilis  yielded  the 
butyric  acid  reaction  in  all  cases  and  at  the  same 
time  gave  a  positive  cytodiagnosis.  As  against 
these  results   is   to  be  placed  the  result  with  the 

Table  32. — Analysis  of  the  reactions  with  regard  to  syphilis. 


No. 

of 

cases 

Butyric  acid 
reaction 

Wassermann 
reaction 

Cell  count 

+ 

- 

± 

+ 

- 

± 

+ 

- 

± 

General  paralysis 
and  tabes : 

Syphilis  + 

Syphilis  — 

Other  diseases: 

Syphilis  + 

Syphilis  — 

36 
16 

1 
12 

34 
13 

1 

1 

1 
3 

0 
11 

1 
1 

0 
0 

26 
10 

1 
3 

8 
S 

0 

8 

2 
3 

0 

1 

36 
11 

1 
1 

0 
3 

0 
11 

0 

2 

0 
0 

Wassermann  reaction,  which  was  positive  in  50 
per  cent,  of  the  cases  examined.  The  cerebro- 
spinal fluid  obtained  from  cases  of  general  paralysis 
gave  positive  butyric  acid  reaction  in  90  per  cent., 
positive  cytodiagnosis  in  91  per  cent.,  and  positive 
Wassermann  reaction  in  73  per  cent,  of  those  ex- 
amined. The  cerebrospinal  fluid  from  cases  of  tabes 
dorsalis  gave  positive  butyric  acid  reaction  and  cyto- 
diagnosis in  all,  or  100  per  cent.,  and  positive  Wasser- 
mann reactions  in  53  per  cent,  of  those  examined. 


230  SERUM  DIAGNOSIS  OF  SYPHILIS. 

Finally,  the  cerebrospinal  fluid  obtained  from  patients 
suffering  with  various  forms  of  psychosis  in  whom 
a  syphilitic  history  was  excludable,  or  at  least  not 
obtained,  gave  positive  butyric  acid  reactions  and 
cytodiagnosis  in  2.8  per  cent,  and  positive  Wasser- 
mann  reactions  in  13  per  cent,  of  those  examined. 

From  the  above  statement  it  becomes  at  once  evi- 
dent that  the  butyric  acid  reaction  runs  parallel  with 
the  cytodiagnosis  in  cases  of  parasyphilitic  disease, 
and  is  especially  reliable  as  an  indicator  of  that  con- 
dition, with  the  cytodiagnosis.  Moreover,  the  results 
of  the  examination  of  the  cerebrospinal  fluid  of  cases 
of  secondary  and  tertiary  syphilis,  in  which  there  are 
no  special  lesions  of  the  central  nervous  system,  in- 
dicate through  the  feeble  reaction  obtained  that  a 
protein  increase  in  the  fluid  is  not  necessarily  associ- 
ated with  an  increase  and  change  in  the  number  and 
quality  of  the  cells  contained  in  the  fluid.  In  other 
words,  the  butyric  acid  reaction  not  only  detects  the 
changes  in  the  fluid  associated  with  parasyphilitic 
disease  and  with  direct  syphilitic  lesions  of  the  central 
nervous  system,  but  it  also  indicates  the  existence  of 
a  general  syphilitic  state  of  infection,  which  cannot 
be  detected  by  the  means  of  cytodiagnosis. 

The  butyric  acid  reaction,  as  stated,  is  about 
parallel  in  results  with  cytodiagnosis,  but  is  not 
parallel  with  the  results  of  the  Wassermann  reaction. 


SERUM  DIAGNOSIS  OF  SYPHILIS.  231 

In  cases  with  established  syphihtic  history,  the  butyric 
acid  reaction  gives  a  higher  percentage  of  positive 
result  than  the  Wassermann  reaction.  It  is  therefore 
somewhat  confusing  to  find  that  in  nonsyphihtic 
forms  of  psychosis,  the  percentage  of  positive  result 
of  the  Wassermann  reaction  exceeds  that  of  the 
butyric  acid  test  or  cytodiagnosis.  This  discrepancy 
has  not  been  cleared  up,  and  calls  for  further  study. 
Whether  or  not  it  has  to  do  with  the  existence  of  an 
independent  constituent  similar  to  syphilitic  "  anti- 
body "  in  the  cerebrospinal  fluid  in  these  cases  needs 
determination. 

It  is  obvious  that  the  butyric  acid  test  is  a  useful 
addition  to  our  diagnostic  methods  in  the  detec- 
tion of  parasyphilitic  diseases  of  the  central  ner- 
vous system,  and  of  cerebrospinal  syphilis.  It  is,  of 
course,  desirable  to  confirm  as  often  as  possible  the 
indications  of  the  reactions  by  post-mortem  examina- 
tions. Of  the  series  which  we  examined,  17  cases 
have  come  to  autopsy.  Of  these,  15  had  given  posi- 
tive butyric  acid  tests,  14  having  been  diagnosed  as 
cases  of  general  paralysis,  and  one  as  a  case  of  cere- 
bral syphilis.  Two  had  given  negative  tests.  The 
autopsy  findings  were  in  complete  agreement  with 
the  indications  of  the  test. 

In  a  series  of  investigations  recently  conducted  at 
the  Kings  Park  State  Hospital,  New  York,  Rosanoff , 


SERUM  DIAGNOSIS  OF  SYPHILIS. 


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Syphilis  ascertained  in ... . 

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.1 

In 

;5 

•pauiniBxa  aaaM 
ifaqi  qorqAi  joj  8abiiDBaj  jua 
-jagip  jnoj  aqj  jo  uo'ijBuiqraoo 
JO  apom  pnB  iJouanb'ajj  aqj  0% 
SaipjoasB  8asB3  jo  Sajdnojf) 

+ 

-H 

1 

cS 

•sjsa 
paur 
-B3a* 
-jnac 

B^qo*  s 
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>jad  pi 

nodsa. 

nOTJOB 
AljlSOC 

iB',»qi 

[100   UI 
3J    aAT) 

[  JO  aSB 
anjsr 

SERUM  DIAGNOSIS  OF  SYPHILIS.  233 

Wiseman,  and  the  writer  confirmed  and  extended  the 
observations  of  Moore  and  the  writer  referred  to 
above.  In  all  413  cases  have  been  examined  from 
the  standpoints  of  the  Wassermann  reaction,  butyric 
acid  test,  and  cytodiagnosis.  Of  this  total  number 
252  cases  were  available  for  the  four  tests  simul- 
taneously (the  Wassermann  reaction  in  serum  and 
in  cerebrospinal  fluid,  butyric  acid  test,  and  cytodiag- 
nosis), while  in  the  remaining  161  cases  the  examina- 
tion of  the  spinal  fluid  was  either  incomplete  or  not 
undertaken.  The  Wassermann  reaction  was  done  by 
the  writer's  system  ^  and  is  designated  in  the  tables  as 
W.-N.  In  Table  33  the  results  obtained  with  general 
paralysis  are  presented.  It  is  interesting  to  notice 
that  the  increase  of  protein  and  of  lymphocytes  in 
cerebrospinal  fluid  of  these  cases  is  distinctly  more 
constant  than  the  presence  of  the  Wassermann  re- 
action. This  last  reaction  was  more  frequently  met 
with  in  the  spinal  fluid  than  in  the  serum.  Note- 
worthy is  it  also  that  the  four  reactions  are  present 
in  70  per  cent,  of  the  cases  examined  and  that  all 
gave  positive  reactions  to  at  least  two  difl'erent  tests. 
This  simultaneous  presence  of  positive  reactions  to 
more  than  two  difl'erent  tests  is  quite  characteristic 
of  parasyphilitic  conditions,  for  it  is  very  seldom  that 
other  forms  of  psychoses  give  plural  positive  re- 

*  See  Chapter  VII,  page  50. 


234 


SERUM  DIAGNOSIS  OF  SYPHILIS. 


actions  without  a  definite  syphilitic  infection.     In 
Tables   33,   34,    35,    36,    37,   I   present   the   results 

Table  34. 


Serum 


W.-N. 


Cerebrospinal  fluid 


W-N.        B.         Cells 


00^ 

m  O 

o  m  a 


Remarks 


Dementia  prsecox 
71  cases 


+ 


45 
4 
9 
2 
3 
2 
1 
4 
1 


50  = 

70% 


64  = 

88.8% 


65  = 

91.5% 


71  = 
100% 


71 


7  = 
10% 


4  = 
6.6% 


6  = 

8.5% 


I        I      GO 

"0.2 

V  S  u 
S  o  2 

O      Q      «H 

es  O 

•a  o£ 

U    V 
M   >  ai 

.■tn    " 

CO    m   V 


u  S  § 

•^2  2 

d  fl  3     . 

9  S  V  « 

«  «  &  S  2 

.S     .  EO  .U  


+ 


14  = 

20% 


3  = 

4.6% 


a,  w>'3 


Mb  O^ 

.  M  °  M  a  S 

'-a:H  §  §  2 


U.2 


'^  .S    CO 

*>       «  O  a) 
w  a*a  4'!7 


Epilepsy  . 
51  cases 


36 
2 
1 
8 
1 
1 
2 


>. 


c^  o 
■'-'...  d 

.2  «  Wi-43        >» 


^  4.*    a; 


38  = 

74% 


48  = 
94% 


49  = 

96% 


51  = 

100% 


51 


6% 


1  = 

2% 


+ 


10  = 

20% 


4% 


4% 


T3 
4) 

o    -    ^ 
>*-•    CO    2 

d. 


^    °T3    O.^   d 

boo  ^  C 


obtained  with  other  forms  of  insanity.    There  are  a 
number  of  cases  giving  solitary  positive  Wassermann 


SERUM  DIAGNOSIS  OF  SYPHILIS.  235 

reaction,  but  scarcely  any  that  gave  positive  cytodiag- 
nosis.    Again,  we  find  that  the  butyric  acid  test  was 

Table  35. 


Serum 

Cerebrospinal  fluid 

"Sc2 
z 

W.-N. 

W.-N. 

B. 

Cells 

Remarks 

Senile  dementia  . 
9  cases 

± 

+ 

+ 

— 

7 
1 
1 

Syphilis  probable 
in  cases  giving  pos- 
itive reactions  for 
the  fixation  and  bu- 

— 

8  = 
88.9% 

8  = 
88.9% 

8  = 
88.9% 

9  = 

100% 

9 

tyric  acid  tests,  but 
diflBcult  to  establish 
with  certainty. 

± 

111% 

0 

0 

0 

None  showed  ple- 
ocytosis. 

+ 

0 

11.1% 

1  = 

11.1% 

0 

Manic  depressive 
insanity,  6  cases 

± 
+ 

+ 

— 

— 

3 

2 
1 

Syphilis  unascer- 
tainable,  but  pos- 
sible in  positive 
cases. 

— 

3 

5 

6 

6 

6 

None  showed  ple- 
ocytosis. 

± 

2 

0 

0 

0 

+ 

1 

1 

0 

0 

Alcoholic  psycho-  ■ 
sis,  4  cases 

± 
+ 

+ 

+ 
d= 

— 

1 
1 
1 

1 

Syphilis  ascer- 
tained in  all  cases 
giving  positive  re- 
actions for  the  fixa- 
tion or  butyric  acid 
tests. 

None  showed  ple- 

— 

1 

3 

1 

4 

4 

± 

2 

0 

2 

0 

ocytosis. 

+ 

1 

1 

1 

0 

positive  in  some  cases  with  syphilitic  histories  and  the 
Wassermann  reaction  in  serum  was  also  positive  in 
these  cases. 


236 


SERUM  DIAGNOSIS  OF  SYPHILIS. 


Samuel  Stern  employed  the  butyric  acid  test  in  a 
large  series  of  psychiatric  conditions  and  obtained  the 
following  results:  General  paresis,  57;  52  positive 
and  4  negative.    Tabes  dorsalis,  3;  3  positive.    One 

Table  36. 


Serum 

Cerebrospinal  fluid 

z 

W.-N. 

W.-N. 

B. 

Cells 

Remarks 

Polyneuritic  psy-  J 
chosis,  7  cases   [ 

+ 

— 

— 

— 

6 
1 

Syphilis  unascertain- 
able  in  the  ease  react- 
ing positively  to  the 
fixation  test  in  serum. 

— 

6 

7 

7 

7 

7 

The  fixation  test  in 
spinal  fluid,  butyric 

+ 

1 

0 

0 

0 

acid  test  and  pleocy- 
tosis  negative  in  all. 

Involution  melan-  / 
cholia,  7  cases    [ 

+ 

— 

— 

— 

6 
1 

Syphilis  unascertain- 
ed in  the  positive  case. 
All  reacted  negatively 

— 

6 

7 

7 

7 

7 

spinal  fluid,  butyric 
acid  test  and  cy  todiag- 

+ 

1 

0 

0 

0 

nosis. 

Paranoic  condi-  f 
tion,  4  cases       [ 

+ 

— 

— 

8 
1 

Syphilis  unascer- 
tainable. 

— 

3 

4 

4 

4 

4 

+ 

1 

0 

0 

0 

Imbecility f 

4  cases                [ 

+ 

— 

— 

— 

2 
2 

Syphilis  unascer- 
tainable. 

— 

2 

4 

4 

4 

4 

+ 

2 

0 

0 

0 

cerebrospinal  syphilis  positive.  Forty-eight  non- 
syphilitic  were  studied  as  controls.  The  controls 
include  the  following  forms  of  psychosis:  Dementia 
prsecox,    manic    depressive,    melancholia,    epilepsy. 


SERUM  DIAGNOSIS  OF  SYPHILIS. 


237 


senility;  toxic  nephritic,  alcoholic  psychosis,  Korsa- 
koff's, postparalytic,  imbecility,  idio-imbecility,  and 
puerperal.  Of  these,  only  two  profound  uremics  and 
one  postparalytic  dementia  were  positive.  Eight 
cadavers;  all  positive. 

Table  37. 


Serum 

Cerebrospinal  fluid 

O    m    g 

W.-N. 

W.-N. 

B. 

Cells 

Remarks 

Infantile  cerebral 
palsy,  4  cases 

— 

— 

— 

— 

4 

Syphilis  unascertain- 
ed. All  reacted  nega- 
tively. 

Arteriosclerotic  de- 
mentia, 7  cases 

— 

— 

— 

— 

7 

Syphilis  unascertain- 
ed. All  reacted  nega- 
tively. 

Brain  tumor 

1  case 

— 

— 

— 

— 

1 

Syphilis  unascertain- 
ed. All  reacted  nega- 
tively. 

Traumatic  psycho- 
sis, 1  case 

— 

— 

— 

— 

1 

Syphilis  unascertain- 
ed. All  reacted  nega- 
tively. 

Unclassified 

82  cases 

+ 

+ 
± 

+ 

± 
+ 
+ 

± 

— 

18 
4 
3 

2 

1 
1 
1 

Syphilis  ascertained 
in  5  cases  reacting  pos- 
itively either   to   the 
fixation  or  to  the  bu- 
tyric acid  test  or  to 
both.     In  other  posi- 
tive cases  it  was  unas- 
certainable  and  unex 
cludable.  None  showed 
pleocytosis. 

McCampbell  and  Rowland  made  a  comparative 
study  of  the  Wassermann  reaction,  butyric  acid  test, 
Ross-Jones  test,  and  cytodiagnosis  on  46  cases  of 
general  paralysis,  5  cases  of  dementia  prsecox,  2  cases 
of  paranoia,  1  case  of  melancholia,  2  cases  of  manic 


238  SERUM  DIAGNOSIS  OF  SYPHILIS. 

depressive  insanity,  3  cases  of  secondary  lues  and 
2  cases  of  tertiary  lues,  including  2  normal  indi- 
viduals. 

Their  results  are  largely  confirmatory  of  ours 
except  that  with  the  Wassermann  reaction  a 
much  higher  percentage  of  positive  reactions  was 
obtained  by  them  with  the  blood  sera  from  para- 
syphilitic  cases.  They  found  exactly  the  same  as 
others  in  regard  to  the  cerebrospinal  fluid,  namely, 
the  percentage  of  positive  reactions  in  the  paret- 
ics was  85.7  per  cent,  for  the  Wassermann  reac- 
tion and  95.6  per  cent,  for  the  butyric  acid  test  and 
cytodiagnosis.  The  Ross-Jones  test  was  slightly 
lower  in  percentage  than  the  butyric  acid  test.  The 
results  obtained  with  the  cerebrospinal  fluids  from 
those  ten  non-parasyphilitic  psychiatric  cases  were  the 
same,  being  uniformly  negative  to  every  test  em- 
ployed. In  the  cerebrospinal  fluid  of  five  syphilitics 
above  mentioned  the  reaction  was  positive  in  all  three 
secondary  cases  for  the  butyric  acid  and  Ross-Jones 
tests,  but  negative  for  the  Wassermann  reaction  and 
cytodiagnosis,  and  in  the  two  tertiary  cases  one  posi- 
tive and  one  doubtful  for  the  butyric  acid  and  one 
positive  and  one  negative  for  the  Ross-Jones  tests, 
while  the  Wassermann  test  and  cytodiagnosis  were 
totally  negative.  The  sera  of  these  syphilitic  cases 
all  gave  positive  Wassermann  reaction  as  would  be 


SERUM  DIAGNOSIS  OF  SYPHILIS.  239 

expected.  Finally  no  positive  reaction  was  obtained 
with  the  cerebrospinal  fluid  or  serum  of  the  two 
normal  persons. 

BUTYRIC    ACID    REACTION    IN    GENERAL    DISEASES. 

That  the  butyric  acid  reaction  will  serve  a  useful 
purpose  in  the  diagnosis  of  nervous  and  mental  dis- 
eases has  not  only  been  indicated  by  my  own  studies  in 
conjunction  with  Dr.  Moore,  but  by  several  reports 
from  other  sources,  in  which  the  reaction  has  been 
applied.  It  is,  however,  necessary  that  the  limit  of 
the  test  should  be  precisely  defined,  which  can  be  done 
by  subjecting  the  cerebrospinal  fluid  from  a  large 
number  of  general  diseases  to  the  action  of  the  test. 
It  wiU  require  such  a  wide  study  to  determine  whether 
or  not  the  reaction  is  either  specific  or  special  for 
syphilitic  disease  in  a  manner  to  render  it  useful  for 
diagnosis.  It  can,  of  course,  be  predicted  that  in  all 
cases  in  which  there  is  increase  in  protein,  and  par- 
ticularly in  the  globulin  fraction  of  the  cerebrospinal 
fluid,  the  reaction  will  be  given.  Now  there  are  other 
diseases  which  produce  this  increase  of  protein  in  the 
cerebrospinal  fluid,  and  they  would  naturally  yield 
the  reaction.  The  chief  if  not  the  sole  diseases  other 
than  syphilis  in  some  of  its  stages,  associated  with 
this  exudative  inflammatory  condition,  are  the  acute 
infections  of  the  cerebrospinal  meninges.  Thus  in 
all  acute  and  subacute  inflammations  of  the  meninges 


240  SERUM  DIAGNOSIS  OF  SYPHILIS. 

a  positive  reaction  is  obtained.  Luckily,  there  is  no 
difficulty  whatever  likely  to  be  experienced  in  the  sepa- 
ration of  this  class  of  cases,  as  has  already  been  men- 
tioned (see  page  157) .  The  only  example  of  inflam- 
mation which  might  be  confused  with  a  syphilitic 
affection  is  tubercular  meningitis,  because,  in  this 
affection,  the  fluid  is  clear  and  contains  an  excessive 
number  of  mononuclear  cells.  But  this  affection  is 
distinguishable  readily  not  only  by  the  chnical  history, 
but  also  by  the  presence  of  the  tubercle  bacilli. 

Should,  however,  cases  arise  in  which  there  re- 
mains some  doubt,  this  can  be  eliminated  readily  by 
invoking  the  aid  of  the  Wassermann  reaction,  in  one 
of  its  forms.  The  cerebrospinal  fluid  in  typhoid  fever 
and  pneumonia,  independent  of  the  acute  inflamma- 
tions of  the  meninges  which  sometimes  attend  these 
diseases,  does  not  yield  the  butyric  acid  reaction. 

The  butyric  acid  reaction  will,  I  believe,  suffice  to 
distinguish  normal  from  pathological  cerebrospinal 
fluid,  and  especially  that  form  of  pathological  fluid 
which  is  altered  through  an  increase  in  its  protein 
constituent.  It  may  therefore  prove  apphcable  to 
some  of  the  ill-defined  inflammatory  conditions  of 
the  meninges — such,  for  example,  as  the  so-caUed 
serous  meningitis,  in  which  condition  the  micro-organ- 
isms and  inflammatory  cells  are  not,  as  a  rule,  de- 
monstrable.   If  the  excessive  serous  exudation  differs 


SERUM  DIAGNOSIS  OF  SYPHILIS. 


241 


from  the  normal  cerebrospinal  fluid  in  the  manner 
which  is  characteristic  of  inflammatory  exudates,  the 
butyric  acid  reaction  would  be  obtainable.  It  also 
suggests  itself  that  the  reaction  would  be  with  profit 


Table  38. —  The  butyric  acid  reaction  in  general  diseases. 

Cases 

No. 

of 

cases 

Butyric  acid 
reaction 

Wassermann 
reaction 

+ 

- 

± 

+ 

- 

± 

Diseases  of  the  meninges: 
Epidemic  cerebrospinal  meningitis . 
Pneumococcal  meningitis 

14 
6 
1 

30 

2 

14 
6 
1 

30 

2 

0 
0 
0 
0 
0 

0 
0 
0 
0 
0 

0 
0 
0 
0 

1 

14 
6 
1 

80 
1 

0 
0 

Influenzal  meningitis 

0 

Tubercular  meningitis 

0 

Hydrocephalus  externus 

0 

53 

53 

0 

0 

1 

62 

0 

Diseases  without  meningeal  involve- 
ment: 
Typhoid  fever 

1 
4 
1 
2 
1 
2 
1 

12 

0 
0 
0 
0 
0 
0 
0 

0 

1 

4 

1 
2 
1 

2 
1 

11 

0 
0 
0 
0 
0 
0 
0 

1 

0 
0 
0 
0 
0 
0 
0 

0 

1 

4 

1 
2 
1 
2 
1 

10 

0 

Pneumonia 

0 

Pulmonary  tuberculosis 

0 

Enterocolitis 

0 

Rachitis 

0 

Uraemia 

0 

Septicaemia 

0 

Miscellaneous  without  nervous  in- 
volvement   

2 

24 

0 

23 

1 

0 

22 

2 

invoked  in  certain  cases  of  tubercular  meningitis  in 
which  tubercle  bacilli  are  not  readily  demonstrable. 
A  positive  reaction  with  butyric  acid  will,  of  course, 
be  given  by  the  cerebrospinal  fluid  provided  there  is 
tubercular  inflammation,  and  the  same  fluid  will  be 


17 


SERUM  DIAGNOSIS  OF  SYPHILIS. 

negative  to  the  Wassermann  test.  Hence  the  two 
tests,  together  with  the  clinical  history,  may  lead  to 
a  provisional  diagnosis  at  a  time  when  the  tubercle 
bacilli  have  not  yet  been  discovered  and  animal  in- 
oculations have  not  yet  had  time  to  declare  the  nature 
of  the  disease. 

BLOOD-SERUM. 

I  have  subjected  about  300  specimens  of 
blood-serum,  taken  from  cases  of  syphilis,  from 
normal  persons,  and  from  persons  suffering  with 
other  diseases  than  syphilis,  to  the  globulin  esti- 
mation by  means  of  the  butyric  acid  precipitation  and 
by  direct  weighing.  According  to  my  observations, 
the  globulin  content  of  normal  blood-serum  varies 
from  0.120  to  0.150  gram  per  0.5  c.c.  of  the  serum, 
weighed  in  the  moist  condition.  The  weight  of  the 
dry  globulin  is  about  one-eighth  to  one-ninth  of  that 
of  the  moist  specimens.  The  blood-serum  which  con- 
tains normal  globulin  content,  prepared  in  the  man- 
ner described  (see  page  225),  does  not  give  a  positive 
butyric  acid  reaction. 

In  cases  of  untreated  and  manifest  secondary  and 
tertiary  syphihs,  the  globulin  content  of  the  serum, 
weighed  in  the  moist  condition,  varies  from  0.200  to 
0.350  gram  per  0.5  c.c.  In  the  primary  stages  of 
syphilis  the  increase  in  globuUn  is  less  great,  but  is 
still  sufficiently  pronounced  to  give  the  butyric  acid 


SERUM  DIAGNOSIS  OF  SYPHILIS.  243 

reaction.  In  cases  of  latent  syphilis,  the  globulin 
content  rarely  exceeds  0.200  gram  and  may  be 
somewhat  less,  but  is  also  sufficiently  increased  to 
give  the  reactions.  On  the  other  hand,  cases  of 
syphilis  which  have  been  well  treated  and  have  not 
exhibited  symptoms  for  many  years,  showed  no  in- 
crease in  the  globulin  content  over  that  of  normal 
individuals. 

I  have  therefore  studied  cases  of  syphilis  in  prog- 
ress of  treatment,  and  have  found  that,  as  the  treat- 
ment progresses  and  the  symptoms  disappear,  the 
reaction  becomes  less  and  less  pronounced,  and  that 
the  globulin  tends  correspondingly  to  approach  closer 
and  closer  the  normal  quantity.  However,  the  butyric 
acid  reaction  does  not  entirely  disappear  from  the 
serum  for  many  months  after  treatment  has  been 
carried  on  and  the  obvious  symptoms  have  entirely 
disappeared.  As  a  rule,  the  butyric  acid  reaction  dis- 
appears later  than  the  Wassermann  reaction,  and  the 
intensity  of  the  butyric  acid  reaction  does  not  always 
run  parallel  with  that  of  the  Wassermann  reaction. 

I  have,  of  course,  studied  the  blood-serum  derived 
from  persons  suffering  from  diseases  of  a  general 
nature,  in  whom  syphihs  could  be  excluded.  It  is 
important  to  record  that,  in  a  small  number  of  cases 
of  carcinoma  and  tuberculosis,  the  butyric  acid  re- 
action was  obtained,  while  the  Wassermann  reactions 
were  negative.     Similarly,  two  cases  of  Hodgkin's 


SERUM  DIAGNOSIS  OF  SYPHILIS. 

disease  which  I  examined  gave  strong  butyric  acid 
reactions,  but  no  Wassermann  reaction.  It  has 
been  pointed  out  by  Gay  and  Fitzgerald  that 
in  some  acute  infectious  diseases,  such  as  pneu- 
monia and  scarlet  fever,  there  may  occur  an 
increase  in  the  globulin  content  leading  to  a 
positive  butyric  acid  reaction.  With  these  facts  in 
mind,  it  is  not  difficult  to  define  the  Hmit  of  ap- 
plication of  the  butyric  acid  reaction  to  blood-serum. 
It  may  therefore  be  stated  that  the  reaction  is  not 
specific,  and  when  it  is  present  it  does  not  necessarily 
indicate  a  syphilitic  infection.  But,  on  the  other 
hand,  it  can  be  employed  to  establish  or  confirm  a 
deduction  based  upon  the  clinical  history  and  the  re- 
sults of  the  Wassermann  reaction  and  thus  become 
indirectly  of  diagnostic  value.  On  the  other  hand,  a 
negative  result  is  valuable  as  eoocluding  a  syphilitic 
infection.  In  this  respect  the  reaction  has  advantages 
over  the  Wassermann  reaction,  in  which  a  negative 
result  is  not  always  reliable  as  indicating  absence  of 
syphilitic  infection. 


GLOSSARY. 

Agglutination. 

Clumping  of  bacteria  or  blood  -  corpuscles  by  specific  agglutinins. 
For  corpuscles  the  term  hsemagglutination  is  often  used. 

Agglutinins. 

A  single  or  repeated  injection  of  bacteria  or  foreign  blood  corpuscles 
into  an  animal  is  followed  by  the  development  of  a  new  property  in 
the  serum  of  that  animal.  This  serum,  when  deprived  of  its  own  com- 
plement either  by  inactivation  or  by  dilution,  is  capable  of  clumping 
in  test  tube  the  bacteria  or  blood  corpuscles  employed  for  immuniza- 
tion. This  phenomenon  is  called  agglutination  and  is  ascribed  to  the 
reaction  product  designated  agglutinin.  Its  nature  is  not  known  ex- 
cept that  it  is  found  in  the  protein  fraction  of  serum  and  resists  the 
temperature  of  56°  C.  Agglutinins  may  be  found  in  some  normal 
sera  in  varying  quantity.  Agglutinins  being  antibodies  are  specific, 
and  can  be  absorbed  by  bacteria  or  blood-corpuscles. 

Alexin. 

First  introduced  by  Hans  Buchner,  adopted  by  Bordet;  is  now  synony- 
mous with  complement  of  Ehrlich  and  cytase  of  Metchnikotf.  See 
Complement.  Buchner's  idea  of  alexin  is  not  identical  with  that  of 
Bordet,  and  the  term  was  used  to  designate  bacteriolysins  and  haemo- 
lysins,  but  not  complement  of  Ehrlich  or  alexin  of  Bordet.  The  term 
alexin  to-day  is  used  in  Bordet's  sense  but  not  in  Buchner's. 

Amboceptor. 

Introduced  by  Ehrlich;  is  synonymous  with  Fixateur  of  Metchnikoff, 
Substance  sensibilisatrice  of  Bordet,  Preparator  of  Max  Gruber,  and 
Copula  of  P.  Th.  Miiller.  Arnboceptor  is  one  of  the  two  active  prin- 
ciples necessary  to  cause  Haemolysis,  Bacteriolysis,  or  any  other 
cytolysis  caused  by  serum,  the  other  active  principle  being  comple- 
ment. Amboceptor  retains  its  activity  after  the  serum  is  heated  to 
from  55°  to  56°  C.  for  30  minutes,  while  complement  is  destroyed  at 
that  temperature.  Amboceptor,  as  well  as  complement,  is  present  in 
the  coagulable  protein  fraction  of  serum.  Amboceptor  may  be  present 
in  any  normal  serum,  and  can  be  produced  in  the  serum  of  an  animal 
by  injecting  repeatedly  the  cells  for  which  it  has  no  amboceptor.  The 
amboceptor  normally  present  is  called  natural  amboceptor  and  that 
which  is  produced  by  means  of  repeated  injections  of  foreign  cells  is 
called  immune  amboceptor.  The  amboceptor  capable  of  causing 
haemolysis  (in  presence  of  complement,  of  course)  is  called  haemolytic 
amboceptor,  while  that  which  is  capable  of  dissolving  bacteria  is 
called  bacteriolytic  amboceptor.  A  few  writers  use  the  simple  terms 
of  haemolysin  or  bacteriolysin  instead  of  haemolytic  or  bacteriolytic 
amboceptor.  Amboceptors  are  capable  of  producing  anti-ambocep- 
tors  when  injected  into  a  susceptible  animal. 

245 


246  GLOSSARY. 

Antibodies. 

A  general  term  applied  to  a  group  of  reaction  products  arising  from 
single  or  repeated  administrations  of  antigens  to  a  suitable  animal. 
Immune  body  is  a  synonym  of  antibody.  Among  antibodies  we  may 
enumerate  hsemolytic  amboceptors,  bacteriolytic  amboceptors,  other 
cytolytic  amboceptors,  precipitins,  agglutinins,  antitoxins,  antivenins, 
antiricin,  antiabrin,  etc.  Antibodies  possess  specific  aflBnity  for  the 
antigens  which  are  used  for  their  production.  Certain  antibodies  such 
as  agglutinins,  amboceptors,  antitoxins,  or  antihsemolysins  may  be 
normally  present  in  certain  sera  in  small  amount.  A  group  of  anti- 
bodies is  capable  of  producing  antibodies  when  injected  into  another 
animal,  thus  forming  anti-antibodies. 

Anticomplementary  action. 

Substances  possessing  the  power  of  reducing  or  removing  totally  the 
action  of  complements  are  said  to  be  anticomplementary.  Most 
acids,  alkalies,  and  certain  salts  have  anticomplementary  action.  In 
certain  sera  there  are  often  certain  principles  possessing  anticomple- 
mentary properties.  Human  serum  gradually  acquires  this  action 
on  standing.  Repeated  injections  of  fresh  serum  into  an  animal  of 
another  species  is  followed  by  the  appearance  of  anticomplements 
(Ehrlich  and  Morgenroth),  while  Gay  considers  it  as  an  example  of 
complement-fixation  by  specific  precipitate. 

Antigens. 

A  general  term  applied  to  a  group  of  substances  capable  of  producing 
specific  antibodies  administered  once  or  repeatedly,  usually  by  injec- 
tion, to  a  suitable  animal.  For  example,  bacteria,  blood-corpuscles, 
and  certain  somatic  cells  are  antigens  because  they  produce  specific 
antibodies  called  amboceptors  and  agglutinins.  Blood-serum,  milk 
or  bacterial  extracts  are  also  antigens,  because  they  produce  anti- 
bodies called  precipitins,  each  being  specific  for  the  substance  em- 
ployed for  its  production.  On  the  other  hand,  most  inorganic  or 
organic  substances  with  definite  chemical  structure  are  not  antigens, 
because  their  introduction  is  not  followed  by  the  formation  of  antago- 
nistic substances  (antibodies)  in  the  body.  Repeated  administra- 
tions of  various  alkaloids  render  the  organism  gradually  more  resis- 
tant to  their  effect,  but  do  not  produce  antibodies,  hence  these  alkaloids 
are  not  antigens.  Diphtheria  toxin,  tetanus  toxin,  rioin,  abrin,  snake 
venoms,  are  antigens  and  their  injections  are  followed  by  specific 
antitoxins,  as  is  well  known. 

Bacteriolysins. 

Active  principles  in  blood-serum  capable  of  dissolving  bacteria,  con- 
sist of  specific  bacteriolytic  amboceptors  and  complement.  Analogous 
to  hsemolysins  and  cytolysins  in  general. 

Bacteriolysis. 

Dissolution  of  bacteria  by  immune  or  normal  sera.  It  is  caused  by 
specific  bacteriolytic  amboceptors  and  complement.  Analogous  to 
haemolysis. 


GLOSSARY.  247 

Bacteriotropins. 

Introduced  by  Neufeld;  are  active  principles  of  certain  immune  sera 
inducing  phagocytosis.  Their  action  is  on  the  bacteria  but  not  on  the 
phagocytes.    They  are  thermostable. 

Complement. 

Introduced  by  Ehrlich;  is  synonymous  with  Metchnikoff's  cytase 
and  Bordet's  alexin.  By  the  term  complement  one  understands  one  of 
the  two  active  principles  concerned  in  Haemolysis,  Bacteriolysis,  and 
other  instances  of  serum  cytolysis.  The  other  principle  is  called  am- 
boceptor, which  is  incapable  of  causing  dissolution  of  cells  without  the 
first,  hence  the  term  complement  is  applied  to  it.  Complement  is 
normally  present  in  all  sera  freshly  drawn  from  the  body,  but  disap- 
pears gradually  on  standing  or  is  completely  destroyed  at  from  56°  to 
66°  C.  in  about  thirty  minutes.  Complement  of  one  species  is  not 
identical  in  its  action  with  that  of  other  species. 

Complement  deflection. 

Synonymous  with  complement  deviation. 

Complement  deviation. 

Synonymous  with  deflection;  originates  from  a  German  term  Ablen- 
kung,  introduced  by  Neisser.  Complement  deviation  is  identical  with 
Komplementablenkung  of  the  Germans,  and  fixation  of  alexin  of  the 
French.  By  the  deviation  of  complement  one  understands  that  com- 
plement is  fixed  by  the  antigen-antibody  combination  and  is  made 
unavailable  for  a  second  set  of  antigen-antibody  combination  to 
complete  a  reaction  in  which  complement  is  essential.  This  second  set 
may  be  a  hsemolytic  or  a  bacteriolytic  system.  See  illustrations  on 
pages  22  and  23. 

Complement  fixation. 

Synonymous  with  complement  deviation. 

Complementoids. 

Modified  complements  in  which  the  zymotoxic  group  is  destroyed 
without  losing  their  binding  property  with  amboceptors.  Complement- 
oids are  formed  at  56°  C. 

Complementophilic  group. 

Atom-complex  of  amboceptor  on  which  complement  anchors.  This 
complex  remains  inactive  until  the  cytophilic  group  (another  atom- 
complex)  of  the  amboceptor  joins  with  the  receptor  of  the  cell. 

Copula. 

Synonymous  with  amboceptor. 

Cytase. 

Introduced  by  Metchnikoff;  is  synonymous  with  alexin  of  Bordet  and 
complement  of  Ehrlich.     See  Complement. 


248  GLOSSARY. 

Cytolysins. 

Active  substances  in  blood-serum  consisting  of  specific  cytolytic  am- 
boceptors and  complement. 

Cytolysis. 

Dissolution  of  cells  by  specific  amboceptors  and  complement.  In 
case  of  blood-corpuscles  the  term  haemolysis  is  used  and  for  bacteria 
the  term  bacteriolysis  is  used. 

Cytophilic  group. 

Atom-complex  of  amboceptor  with  which  the  receptor  of  a  cell  com- 
bines. Thus  an  amboceptor  possesses  two  atom-complexes,  one  for 
the  complement  and  the  other  for  the  receptor  of  the  cell. 

Endotoxin. 

Toxic  constituents  of  bacterial  cells. 

Fixateur. 

Metchnikoff's  term  for  Amboceptor  of  Ehrlich  and  Substance  sensi- 
bilisatrice  of  Bordet.     See  Amboceptor. 

Haemolysins. 

Any  substance  capable  of  causing  haemolysis  may  be  called  an  heem- 
olysin,  but  its  use  is  restricted  to  the  biological  products  of  unknown 
chemical  constitution,  especially  the  blood-serum,  or  often  the  ambo- 
ceptor of  the  serum. 

Haemolysis. 

Dissolution  of  blood-corpuscles  by  various  forces,  setting  the  hsemo- 

flobin  free  into  the  medium  in  which  the  corpuscles  are  suspended. 
)istilled  water,  freezing  and  thawing,  temperature  of  about  55°  C.  for 
30  minutes,  etc.,  are  physical  agents  which  cause  haemolysis.  Acids, 
alkalies,  and  certain  salts  can  cause  haemolysis  in  proper  concentra- 
tions. Of  these  chemicals  may  be  mentioned  most  organic  acids, 
mineral  acids,  all  alkalies,  bile  salts,  bichloride  of  mercury,  soaps. 
Of  biological  origin  may  be  mentioned  certain  glycosides  such  as 
saponin,  solanin,  etc.,  certain  bacterial  cultures  such  as  those  of  staph- 
ylococcus, vibrios,  megatherium,  tetanus  bacillus,  etc.;  certain 
animal  venoms  such  as  those  of  snakes,  bees,  spiders,  etc.  The  haemo- 
lytic  process  caused  by  these  different  agents  is  different  according 
to  the  nature  of  the  hsemolytic  forces,  but  they  attack  the  corpuscles 
more  or  less  directly.  Haemolysis  by  serum  is,  however,  somewhat 
different  from  that  caused  by  the  various  forces  just  mentioned. 
Thus,  haemolysis  by  fresh  alien  serum  is  caused  by  two  distinct  groups 
of  substances,  both  contained  in  blood-serum.  One  is  called  com- 
plement and  the  other  amboceptor.  The  one  is  inactive  without  the 
other.  Serum  haemolysis  forms  the  basis  of  many  interesting  phe- 
nomena, the  serum  diagnosis  of  syphilis  being  one  of  these. 

Haemolytic  amboceptors. 
See  under  Amboceptors. 


GLOSSARY.  249 

Haptins. 

Introduced  by  Ehrlich;  synonymous  with  antibodies,  except  in  some- 
what broader  sense. 

Haptophore  group. 

The  atom-complex  of  complement  which  has  the  power  of  anchoring 
on  the  complementophilic  group  of  amboceptor,  thus  uniting  com- 
plement with  the  cell  through  the  intermediation  of  amboceptor. 

Immune  bodies. 

Synonymous  with  antibodies. 

Inactivation. 

Fresh  serum  containing  both  amboceptor  and  complement,  becomes 
inactive  when  heated  to  from  55°  to  56°  C.  for  about  30  minutes  be- 
cause of  the  destruction  of  complement.  This  process  is  called  inac- 
tivation, and  the  heated  serum  is  called  inactivated  serum.  Ambo- 
ceptor is  not  affected  materially  by  the  process. 

Inter-body. 

Ehrlich  used  the  term  Zwischenkorper  before  he  introduced  the  term 
Amboceptor,  and  its  English  version  is  Inter-body  (Bolduan)  or  Inter- 
mediary body  (Flexner  and  Noguchi). 

Intermediary  body. 

Synonymous  with  Inter-body;  an  English  translation  of  Ehrlich's 
Zwischenkorper.     Identical  with  amboceptor. 

Iso-agglutinin. 

Blood-serum  of  an  animal  usually  does  not  contain  agglutinins  for 
the  blood-corpuscles  of  another  animal  of  the  same  species,  but  in 
some  instances  agglutination  may  occur  and  is  due  to  the  substances 
called  iso-agglutinins. 

Iso-hsmolysin. 

Blood-serum  of  one  .animal  usually  does  not  hsemolyse  the  blood-cor- 
puscles of  another  animal  of  the  same  species,  but  in  some  instances 
haemolysis  may  occur.  This  phenomenon  is  known  as  isohssmolysis 
and  is  caused  by  the  presence  of  iso-hsemolysin.  In  man  this  is  ob- 
served quite  frequently  in  the  serum  of  patients  suffering  from  malig- 
nant tumors. 

Komplementablenkung. 

Synonymous  with  complement  deviation. 

Komplementbindung. 

Synonymous  with  complement  deviation. 

Komplementverankelung. 

Synonymous  with  complement  deviation. 


250  GLOSSARY. 

Komplementoidverstopfung. 

Prevention  of  complement  fixation  on  account  of  interference  on  the 
part  of  complementoid.  In  case  of  heemolysis,  this  causes  inhibition 
of  haemolysis. 

Opsonins. 

Introduced  by  A.  E.  Wright;  are  active  substances  of  normal  as  well 
as  immune  sera  causing  pnagocytosis.  Normal  opsonins  are  rendered 
inactive  at  56°  C.  and  seem  to  depend  upon  the  cooperation  of  comple- 
ment.    Immune  opsonins  are  thermostable. 

Pleocytosis. 

Introduced  by  Nonne  and  identical  with  lymphocytosis  in  the  cere- 
brospinal fluid  in  syphilitic  and  parasyphilitic  diseases  of  the  central 
nervous  system. 

Precipitates. 

By  the  term  precipitate  in  immunity  work  is  meant  the  flocculence 
or  clumps  formed  by  mixing  specific  antigen  and  antibody,  such  as 
serum  precipitates,  bacterial  extract  precipitates,  etc. 

Precipitation. 

In  immunity  work  one  understands  by  precipitation  a  clumping 
phenomenon  of  protein  or  protein-like  substances  by  specific  pre- 
cipitins. 

Precipitin. 

In  the  blood-serum  of  an  animal  which  received  repeated  injections 
of  a  solution  of  proteid  matter  there  is  found  a  substance  capable  of 
precipitating  that  proteid  when  mixed  in  a  test  tube.  This  precipitat- 
ing principle  is  called  precipitin,  and  its  action  is  specific;  that  is,  a 
?recipitin  for  human  serum  precipitates  the  latter,  but  no  other  serum, 
recipitins  can  be  produced  in  animals  for  different  proteins,  such  as 
egg  albumin,  serum,  milk,  bacterial  proteins,  etc.  It  is  resistant  to 
the  temperature  of  66°  C.  like  most  immunization  products  and  re- 
mains active  for  a  very  long  time  when  desiccated. 

Precipitinogen. 

A  general  term  occasionally  used  for  the  substances  capable  of  pro- 
ducing precipitins  by  means  of  immunization,  or  repeated  injections 
into  animals. 

Preparator. 

Synonymous  vsdth  Amboceptor. 

Protectin. 

A  term  introduced  by  Noguchi  to  designate  a  substance  (or  substances) 
developing  in  all  blood-sera  on  standing  in  vitro,  and  characterized 
by  its  or  their  effects  in  protecting  blood-corpuscles  against  hsemolytic 
serum.  This  protective  substance  (or  substances)  is  taken  up  by  the 
corpuscles  through  long  contact,  this  property  being  increased  in  sera  of 


GLOSSARY.  251 

certain  animals  after  heating  to  60°  C.  or  a  little  higher.  It  is  similar 
to  the  complementoid  of  Ehrlich  and  Morgenroth,  difFering  only  in 
its  absorbability  by  non-sensitized  cells  and  extractability  by  fat  sol- 
vents such  as  ether,  acetone,  and  alcohol. 

Reactivation. 

The  addition  of  complement  to  an  inactivated  serum  restores  its  lytic 
activity,  and  the  process  is  called  reactivation. 

Receptors. 

Constituents  of  the  cell  uniting  with  amboceptors  or  any  other  anti- 
bodies or  toxins.  The  presence  or  absence  of  receptors  determines 
whether  the  cell  is  susceptible  to  a  given  amboceptor  or  toxin,  or  not. 

Sensibilisation. 

Synonymous  with  sensitization  and  is  caused  by  allowing  amboceptor 
to  act  on  cells. 

Sensitization. 

When  a  cell  is  acted  upon  by  specific  amboceptor  it  becomes  sensitive 
to  the  dissolving  action  of  complement.  This  process  of  rendering  a 
cell  sensitive  is  called  sensitization.    In  French  it  is  sensibilisation. 

Sensitizer. 

Synonymous  with  Amboceptor  of  Bhrlich  and  Bordet's  Substance 
sensibilisatrice. 

Sensitizing  substance. 

Synonymous  with  Amboceptor. 

Stimulin. 

Introduced  by  Metchnikoff;  is  an  active  principle  of  serum  favoring 
phagocytosis.  Metchnikoff  thought  it  due  to  the  stimulation  of 
phagocytes,  but  it  is  probably  identical  with  the  opsonins  of  Wright. 

Substance  sensibilisatrice. 

Introduced  by  Bordet;  is  synonymous  with  Ehrlich's  Amboceptor, 
Metchnikoff's  Fixateur,  Gruber's  Preparator,  or  P.  Th.  MuUer's 
Copula.  Bordet  often  uses.the  term  "sensibilisatrice."  See  Ambo- 
ceptor. 

Thermolabile. 

Complement  loses  its  activity  at  about  55°  to  66°  C.  in  about  80 
minutes  and  is  called  thermolabile. 

Thermostable. 

Amboceptor  remains  stiU  active  after  the  serum  containing  it  is  heated 
at  55°  to  56°  C.  for  SO  minutes,  and  is  said  to  be  thermostable. 


252  GLOSSARY. 

Toxins. 

A  general  term  for  a  group  of  substances  mostly  of  bacterial  elabora- 
tion, possessing  a  powerful  toxicity  and  one  or  more  of  the  following 
characteristics.  Thermolability,  incubation  period  for  action,  unknown 
chemical  constitution,  difficulty  in  separating  from  protein  molecule, 
capability  of  producing  antibodies.  Best  known  examples  are  diph- 
theria toxin  and  tetanus  toxin,  both  of  which  belong  to  true  toxins  and 
are  of  extracellular  origin.  The  toxic  principles  of  cholera  vibrio,  menin- 
gococcus, gonococcus,  typhoid  bacillus,  dysentery  bacillus  are  chiefly 
contained  m  the  cell-body  and  are  called  endotoxins.  Tuberculin  is 
an  atypical,  extracellular  toxin.  Toxalbumins  of  higher  plants  and 
toxic  secretions  of  snakes,  spiders,  and  bees  resemble  bacterial  toxins 
in  many  respects. 

Toxoids. 

Ehrlich  modified  various  toxins  by  chemicals  in  such  a  manner  as  to 
reduce  or  remove  their  toxic  property  without  destroying  their  im- 
munizing property.  They  may  arise  spontaneously  under  certain 
circumstances  and  combine,  like  toxins,  with  antitoxins.  The  modi- 
fied toxins  are  called  toxoids. 

Toxophore  group. 

Analogous  with  zymotoxic  group  of  complement. 

Zymotoxic  group. 

Analogous  with  toxophore  group  of  a  toxin  and  represents  the  active 
dissolving  atom-complex  of  a  complement.  The  destruction  of  this 
group  leads  to  the  formation  of  a  complementoid. 


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20 


290  BIBLIOGRAPHY. 

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INDEX 

FAOB 

Active  serum,  definition  of 3 

use  of,  in  syphilis  serum-reaction 41-47,  99-101 

Adjustability  of  the  Noguchi  system 90-94 

Agglutinins  viewed  as  antibodies 18 

Alcoholic  psychosis 133,  228,  235 

Alexins.     See  Complements. 

Amboceptors,  definition,  occurrence,  and  function  of 1-2,  5 

stability  of 2 

specificity  of 5 

relation  of  amboceptors  to  different  complements 5 

designation  of 5 

natural 6 

immune 6 

isohsemolytic 6 

unit  of 11 

effects  of  multiple  units  of,  on  haemolysis 13 

viewed  as  antibodies 17 

fate  of,  after  haemolysis 15 

hsemolytic  and  bacteriolytic 17 

excess  of,  as  a  disturbing  factor  in  the  complement-fixation  tests  34,  122 

preparation  of,  for  the  Noguchi  system 74-77 

preparation  of,  slips 77-78 

preparation  of,  for  the  Wassermann  system 108 

Antibodies,  definition  and  varieties  of 17-25 

specific  nature  of 18 

behaviors  of  antibody-antigen  combinations  on  complements. ...      20 
methods  of  detection  of  unknown 25 

Anticomplementary:  development  of  anticomplementary  property  in 

human  serum 92 

removal  of  anticomplementary  property  by  inactivation  process. .     92 

Antigens,  definition  and  varieties  of 17-25 

specific  affinities  for  antibodies 18 

method  for  detecting  unknown 19 

fixation  of  complement  by  combinations  of  specific  antigen-anti- 
body       20 

syphilitic  antigens.    See  under  that  heading. 

299 


300  INDEX 

Antisyphilitic,  effect  on  the  Wassermann  reaction  of,  treatments . .   136-158 
effect  on  the  globulin  content  of  blood  serum  of,  treatments. .    221-244 

Aortic  insufficiency 131 

Arsenobenzol,  effect  of,  upon  syphilis 141-158 

Arteriosclerotic  psychosis 133,  237 

Ascitic  fluids 131 

Autopsy,  as  a  means  of  confirming  ante-mortem  finding  by  the  Wasser- 
mann and  the  butyric  acid  reactions 231 

Bacteria,  viewed  as  antigen 17 

Bacteriolysis 1''' 

Bacteriolytic  amboceptors 17 

Banti's  disease 123, 130 

Bauer 39, 42. 46 

Blood  serum,  the  Wassermann  reaction  in 114-135 

Boas 42, 47 

Bordet-Gengou  phenomenon 21-24 

Brain  tumors 125, 133,  237 

Browning 41,  42,  47 

Bruck,  Wassermann,  Neisser  and 27, 37,  46 

Butyric  acid  reaction.    See  Butyric  acid  test. 

Butyric    acid    test,    relation    between  the    Wassermann     and     the 

butyric  acid  tests 221-244 

application  to  cerebrospinal  fluid 223 

in  syphilis  and  parasyphilis 227-239 

in  hereditary  syphilis 228 

in  nonsyphilitic  inflammation  of  meninges 225 

in  normal  spinal  fluid 225 

application  to  blood  serum 225-227 

comparison  of  the  Wassermann  reaction,  cytodiagnosis  and, 

226-227 

in  general  diseases 239 

as  a  quick  means  of  distinguishing  pathological  from  normal 

cerebrospinal  fluids 226 

in  blood  serum 225-227 

post-mortem  confirmation 231 

Cancer.     See  Carcinoma. 

Carcinoma 125, 130 

Cerebrospinal  fluid,  the  Wassermann  reaction  in 121,  122,  127,  227-239 

Cerebrospinal  syphilis 117, 125, 149 

Chronic  arthritis , 123, 131 


INDEX  301 

.  Cirrhosis  of  liver 131 

Complements,  definition,  occurrence  and  function 1-2 

spontaneous  deterioration  of 2 

inactivation  by  heat 4 

designation  of 4 

interchangeability  of  different 4 

in  immune  serum 6 

in  normal  serum 8 

human  complement  against  human  corpuscles 10 

unit  of 12 

fate  of,  after  haemolysis 15 

absorption  or  deviation  of 20-21 

fixability  of  various 21-22 

delicacy  of  complement-fixation  test 21 

preparation  of,  for  complement-fixation  test 72-74, 107 

Complement-deviation  tests.    See  Complement-fixation  test. 

Complement-fixation  test,  general  rule 32-33,  35 

Constitutional  inferiority 133 

Corpuscle-suspension,  for  the  Noguchi  system 56-58 

for  the  Wassermann  system 108 

Cytodiagnosis,  comparison  of  the  Wassermann,  butyric  acid  test  and, 

227-239,  241-242 

Deflection  of  complement.     See  Complement-fixation. 

Dementia  paralytica.     See  General  paralysis. 

Dementia  prsecox 132,  133,  228,  234 

Dementia  senilis.    See  Senile  dementia. 

Detre 27,  47 

Deviation,  of  complement.     See  Complement-fixation. 

Diabetes  mellitus 124, 129, 131 

Diagnostic  value,  of  the  Wassermann  reaction 114-135 

of  the  cytodiagnosis 227-239 

of  the  butyric  acid  reaction 221-244 

Dioxydiamidoarsenobenzol.     See  Arsenobenzol. 

Eczema 124, 127, 131 

Egg-albumin,  viewed  as  antigen 17 

Ehrlich-Hata  arsenobenzol.     See  Arsenobenzol. 
Ehrlich-Hata  "606."     See  Arsenobenzol. 

Endothelioma 130 

Epilepsy 131,  133,  234 

Errors,  in  complement-fixation  tests 90-93 

Erythrocytes,  viewed  as  antigen 17 

Euglobulin,  increase  of,  in  syphilis 221 


302  INDEX 

Extracts,  aqueous,  as  syphilitic  antigen 102-104 

alcoholic,  as  syphilitic  antigen 104-105 

aceton-insoluble  fraction  of  tissue-extracts 106 

Fallacies,  in  complement-fixation  tests 90-94 

Fixability,  of  complements 21-22,  Sl-32 

Fixation,  of  complement 32-33,  35 

General  diseases,  the  cerebrospinal  fluids  in 239 

General  paralysis 121,  125,  228,  229,  231,  232,  236,  237 

General  paresis.     See  General  paralysis. 

Gengou-Bordet  phenomenon 21-25 

Globulin,  abnormal  high  content  in  syphilis  and  parasyphilis  of.    221-222 

Haemolysis 1, 17 

Hsemolytic  amboceptors 17 

Hsemolytic  system 31,  36,  37,  38,  39,  44,  45 

Hecht 39,  42,  46 

Hemiplegia 131 

Hodgkin's  disease 130 

Idiocy 131, 133 

Imbecility 131,  133,  228,  236 

Inactivation,  of  serum 4 

effect  on  syphilitic  serum 95 

effect  on  leprous  serum 98 

Infantile  cerebral  palsy 133,  237 

Insanity,  the  Wassermann  reaction  in 121,  227-239 

Involution  melancholia 133,  236 

Iritis 132 

Isohsemolysins 6 

Keratitis  interstitialis 132 

Landsteiner,  discovery  of  antigenic  property  of  alcoholic  extracts.  ...     29 

method  of  preparing  syphilitic  antigen 105 

Leprosy,  the  Wassermann  reaction  in 128,  129 

Lesser.     See  Michaelis. 

Levaditi,  method  of  preparing  syphilitic  antigen 103 

Lipoids,  as  syphilitic  antigen 79 

Liver,  of  congenital  syphilitic  foetus  as  antigen 27,  29, 102 

Luetin  reaction 167-220 

method  of  preparation  and  application 168-169 

in  rabbits 170 

in  man 172-220 


INDEX  303 

Lupus 127,  130 

Malignant  tumors 125,  130 

Marie.     See  Levaditi. 

Manic  depressive  insanity 133,  228  ,235 

Measles 130 

Meier.     See  Porges. 

Meltzer,  method  of  administration  of  "  606 " 146 

Meningitis,  nonsyphilitic 241 

Mercury,  effect  on  the  Wassermann  reaction  of 136-141 

effect  on  the  globulin  content  of  blood  serum 242,  244 

Metasyphilis.     See  Parasyphilis. 

Michaelis,  method  of  preparing  syphilitic  antigen 105 

Morgenroth,  method  of  preparing  syphilitic  antigen 104 

Miiller.     See  Landsteiner. 

Miiscular  dystrophy 130 

Neisser,  Wassermann,  Bruck 27,  37,  46 

X<}ervous  diseases 124-125,  131-133 

Neurasthenia 130 

Noguchi  method  of  preparing  syphilitic  antigen 79,  106 

Noguchi  system,  of  complement-fixation  test 50-89 

detailed  description  of 79-89 

results  obtained  by 122-133,  139-141, 147-153,  227-241 

adjustability  of 90-94 

apparatus  for 53-54 

inactivated  serum  for 59,  62,  63 

corpuscle  suspension  for 56-58 

patient's  serum 54 

Nonspecificity  of  the  Wassermann  reaction 114 

Optic  atrophy 132 

Paranoiac  conditions 236 

Parasyphilis,  the  Wassermann  reaction  in 121,  227 

Patient's  serum,  for  the  Noguchi  system 54 

for  the  Wassermann  system 108 

inactivation  of,  for  the  Noguchi  system 59,  62,  63 

Phosphatids,  as  a  syphilitic  antigen 79,  106 

Pleocytosis,  or  lymphocytosis  in  psychiatry 227-239,  241-242 

Poliomyelitis,  butyric  acid  test  in 225 

Polyneuritic  psychosis 133,  236 

Porges,  method  of  preparing  syphilitic  antigen 104 

Potzl.     See  Landsteiner. 


304  INDEX 

Precipitins 17 

viewed  as  antibodies 18 

specificity  of 18 

Precipitinogen 20 

Raynaud's  disease 131 

Reactivation  of  serum 4 

Receptor 2 

Relapses  of  syphilis  and  the  Wassermann  reaction 142-153 

Rondoni.     See  Sachs. 

Sachs,  artificial  syphilitic  antigen 106 

Salvarsan.     See  Arsenobenzol. 

Sarcoma 130 

Scarlatina 129, 130 

Scarlet  fever.     See  Scarlatina. 

Schiirmann,  artificial  syphilitic  antigen 107 

Scleroderma 124, 131 

Senile  dementia 132,  133,  235 

Serum,  active  or  fresh 3 

inactivated 4 

inactivation  of 4 

reactivation  of 4 

determination  of  hsemolytic  activity  of 8 

heemolytic  titre  of 8 

determination  of  hsemolytic  activity  of  immune  serum 9 

viewed  as  antigen 17 

collection  of  patient's  serum  for  the  Noguchi  system 54 

use  in  the  Noguchi  system  of  the  inactivated 59,  62,  63 

Serum  diagnosis,  various  forms  of,  for  syphilis 36-49 

Serum  haemolysis,  definition  and  mechanism  of 1-7 

quantitative  facts  about 8-16 

fallacies  in  conclusions  based  on 14 

"606."     See  Arsenobenzol. 

Spastic  hemiplegia 131 

Specific  complement-fixation  test  for  syphilis 159-166 

Spirochsete  pallidum 27 

Stern 39,  46 

Stertz.     See  Morgenroth. 

Suspension  of  blood-corpuscles 56-58, 108 

Syphilitic  antibodies,  methods  for  detecting 26-30 

titration  of 69-72 

effect  of  inactivation  on 95-101 

effect  of  temperature  on 96-99 


INDEX  305 

Syphilitic  antibodies,  rate  of  destruction  by  heat 96-99 

detection  of  specific 159-166 

Syphilitic  antigens,  general 27 

lipoidal  nature  of 29 

syphilitic  tissues  as 27,  29 

for  the  Noguchi  system 79-89 

titration  of 80-88 

preservation  of 88-89 

for  the  Wassermann  system 102-107 

nonspecificity  of 29,  114 

phosphatids  as 79 

artificial 106 

aqueous  organ  extracts  as 102-104 

alcoholic  organ  extracts  as 104-105 

Syphilitic  serum,  titration  of 69-72,  145-150 

effect  of  inactivation  on 95-101 

anticomplementary  action  of  old 92 

Tabes  dorsalis 121,  125 

Thrombo-angiitis  obiterans 125 

Traumatic  psychosis 133,  228,  237 

Treatments,  in  relation  to  the  Wassermann  reaction 114-153 

effect  on  the  globulin  content  of  blood  serum 243-244 

Treponema  pallidum 27 

as  antigen 159 

Tschernogubow 41-42,  47 

Tuberculosis 125, 130,  241 

Unit,  of  amboceptor 11 

of  complement 12 

of  corpuscle  suspension 12 

of  different  factors  in  haemolysis 12 

Ursemic  psychosis 133 

Varicella 130 

Wassermann  method  of  preparing  syphilitic  antigen 102-103 

Wassermann,  Neisser  and  Bruck 27,  37,  46 

Wassermann  reaction,  the  origin  of 1 

diagnostic  value  of 114-135 

specificity  of 114 

in  primary  syphilis 116,  118,  119 

in  secondary  syphilis 116, 119 


306  INDEX 

Wasserman  reaction  in  tertiary  syphilis 116,  119 

in  latent  syphilis 116,  119 

in  hereditary  syphilis 117,  120 

in  cerebrospinal  syphilis 117,  120 

in  parasyphilis 117,  121,  227 

in  various  diseases 122-131 

in  Dermatology 124, 127 

in  Ophthalmology 132 

in  Neurology 133 

in  Gynaecology 135 

in  Psychiatry 133,  227-239 

in  relation  of  the  laws  of  Colles  and  Profeta 135 

Wassermann  system,  complete  description  of 102-113 

Wright,  capsule  for  collection  of  serum 55 

Yamanouchi.     See  Levaditi. 


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